CN116769695A

CN116769695A - Culture medium and method for producing human cells and tissues from teratomas, organoids and embryoid bodies

Info

Publication number: CN116769695A
Application number: CN202211317527.8A
Authority: CN
Inventors: 米格尔·A·埃斯特班; 李文娟; 穆罕默德·阿卜杜尔·马西德; 姜禹; 赖毅维; 骆志伟; 李金秀
Original assignee: Guangzhou Institute of Biomedicine and Health of CAS
Current assignee: Guangzhou Institute of Biomedicine and Health of CAS
Priority date: 2022-03-17
Filing date: 2022-10-26
Publication date: 2023-09-19
Also published as: WO2023173370A1

Abstract

The present invention relates to media and methods for producing human cells and tissues from teratomas, organoids and embryoid bodies. The method of the invention for producing teratomas comprises the steps of transplanting primary, originating, 8clc or heavy primary PSCs into different organs or locations of an immunodeficient animal of interest and feeding the animal; wherein the method of making the primary PSCs/ICLCs comprises the step of culturing primate PSCs in a medium comprising an SAH/PRC/EZH2 inhibitor, an HDAC inhibitor and a WNT/beta-catenin signaling inhibitor; the preparation method of the 8CLCs comprises the steps of culturing primate PSCs or the original PSC/ICLC in a culture medium containing optimized doses of SAH/PRC/EZH2 inhibitor, HDAC inhibitor and WNT/beta-catenin signal inhibitor; the heavy original state PSCs are obtained through induced differentiation of original state PSCs/ICLCs or 8 CLCs.

Description

Culture medium and method for producing human cells and tissues from teratomas, organoids and embryoid bodies

Technical Field

The present invention relates to media and methods for producing human cells and tissues from teratomas, organoids and embryoid bodies.

Background

Human cells with potential therapeutic applications include fully differentiated cells such as T and B lymphocytes, somatic stem/progenitor cells, such as Hematopoietic Stem Cells (HSCs) and hepatic progenitor cells (LPCs). These cells may be isolated from the human body or obtained by differentiation of pluripotent/totipotent stem cells. In general, cells isolated from the human body are functional and do not cause immune rejection when used either by themselves or by HLA-typed suits. However, not all types of cells can be isolated from the human body, and there is a problem in that the number of cells that can be isolated is rare. In addition, isolated cells may exhibit dysfunction and time is required to find a patient with appropriate HLA assignment. Pluripotent/totipotent stem cells can be expanded indefinitely and easily engineered, and autologous and analog (HLA-universal, surface proteins removed to reduce immunogenicity) pluripotent/totipotent stem cells can be used to produce target derivatives. However, the target derivatives produced by pluripotent/totipotent stem cells may not differentiate properly or function.

In order for derivatives of pluripotent/totipotent stem cells to be suitable for clinical treatment, the cells need to achieve the correct cell state and function during differentiation. In this regard, scientists have been striving to develop various in vitro lineage specific differentiation protocols for Pluripotent Stem Cells (PSCs). Some schemes are more effective. For example, induction of differentiation in vitro to generate neural stem cells and use for transplantation has been partially successful. Preclinical experiments are being conducted in animal models, including non-human primates, and several clinical experiments are also underway. However, other protocols (such as HSC and LPC) remain challenging due to limited efficiency and loss of function. The main reasons are as follows: 1) The starting PSCs have epigenetic abnormalities that result from prolonged culture or reprogramming, or limited flexibility in chromatin state; 2) In vitro differentiation protocols fail to completely mimic the differentiation pathway in vivo. Due to the initiation of epigenetic events, primitive stem cells or totipotent like cells may have greater flexibility and differentiation potential than originating stem cells. Furthermore, the in vivo environment can effectively induce PSC differentiation, which is difficult to achieve in vitro. The combination of these two features may contribute to the generation of functionally differentiated cells.

Disclosure of Invention

The first aspect of the present invention provides a method of producing a teratoma, the method comprising bringing into a pristine stateA step of transplanting PSC/ICLC, an original PSC,8CLC or a reprimed PSC into different organs or locations of an immunodeficient animal of interest and raising said animal; wherein the method of preparing the primary PSC/ICLC comprises the step of culturing primate PSC in a medium comprising an SAH/PRC/EZH2 inhibitor, an HDAC inhibitor and a WNT/beta-catenin signal inhibitor; the 8CLC preparation method comprises the steps of culturing primate PSC or the original PSC/ICLC in a medium containing optimized doses of SAH/PRC/EZH2 inhibitor, HDAC inhibitor and WNT/beta-catenin signal inhibitor; the heavy primary state PSC is obtained by induced differentiation of primary state PSC/ICLC or 8 CLC.

In a second aspect, the present invention provides a method for producing an organoid, said method comprising the step of suspension-culturing an original state PSC/ICLC,8CLC or a re-original state PSC, or culturing said original state PSC/ICLC,8CLC or re-original state PSC on a 3D scaffold in a medium that enables differentiation into a target organ; wherein the method for preparing the original PSC/ICLC comprises the step of culturing primate PSC in a medium comprising SAH/PRC/EZH2 inhibitor, HDAC inhibitor and WNT/beta-catenin signal inhibitor; the 8CLC preparation method comprises the steps of culturing primate PSC or primary PSC/ICLC in a medium containing optimized doses of SAH/PRC/EZH2 inhibitor, HDAC inhibitor and WNT/beta-catenin signal inhibitor; the heavy primary state PSC is obtained by inducing differentiation of primary state PSC/ICLC or 8 CLC.

In a third aspect, the present invention provides a method for producing embryoid bodies, comprising the step of culturing the primary state PSC/ICLC,8CLC or the heavy primary state PSC in a medium capable of differentiating it into a target organ; wherein the method for preparing the original PSC/ICLC comprises the step of culturing primate PSC in a medium comprising SAH/PRC/EZH2 inhibitor, HDAC inhibitor and WNT/beta-catenin signal inhibitor; the 8CLC preparation method comprises the steps of culturing primate PSC or primary PSC/ICLC in a medium containing optimized doses of SAH/PRC/EZH2 inhibitor, HDAC inhibitor and WNT/beta-catenin signal inhibitor; the heavy primary state PSC is obtained by inducing differentiation of primary state PSC/ICLC or 8 CLC.

In one or more embodiments, the medium further comprises one or more of L-ascorbic acid or a derivative thereof, a JAK/STAT3 signaling activator, and a MAPK/ERK signaling inhibitor; optionally, the medium is further supplemented with one or more of an ACTIVIN/NODAL signal activator, a ROCK inhibitor, and an extracellular matrix.

In one or more embodiments, the PRC/EZH2 inhibitor or SAH inhibitor is selected from DZNep and CPI-1205; the final concentration of DZNep in the medium is preferably 5-80nM, more preferably 5-50nM; the final concentration of CPI-1205 in the medium is preferably 0.5-5mM, more preferably 1-3mM.

In one or more embodiments, the HDAC inhibitor is selected from TSA, VPA, and NaB; the final concentration of TSA in the medium is preferably 3-30nM, more preferably 3-25nM; the final concentration of VPA in the medium is preferably 0.25-2mM, more preferably 0.5-1.5mM; the NaB concentration in the medium is preferably 0.25-2mM, more preferably 0.5-1.5mM; and/or the final concentration of the WNT/beta-catenin signal inhibitor in the culture medium is 2-8 mu M; preferably, the WNT/beta-catenin signal inhibitor is selected from IWR1 and XAV939.

In one or more embodiments, the final concentration of L-ascorbic acid in the medium is 40-70 μg/mL.

In one or more embodiments, the JAK/STAT3 signal activator is present at a final concentration of 10-50ng/mL; the JAK/STAT3 signal activator is preferably LIF.

In one or more embodiments, the final concentration of the MAPK/ERK signaling inhibitor is 0.5-3. Mu.M; the MAPK/ERK signaling inhibitor is preferably PD0325901.

In one or more embodiments, the final concentration of the ACTIVIN/NODAL signal activator is 10-25ng/mL; preferably, the activator of the ACTIVIN/NODAL signal is selected from ACTIVIN a and NODAL.

In one or more embodiments, the ROCK inhibitor is present at a final concentration of 0.5 to 2 μm; preferably, the ROCK inhibitor is selected from the group consisting of Y27632, thiazovivin (CAS number 1226056-71-8;N-benzyl-2- (pyrimidin-4-ylamino) thiazole-4-carboxamide) and hydroxyfasudil.

In one or more embodiments, the extracellular matrix is present in the medium in an amount of 0.1-0.5% (v/v); preferably, the extracellular matrix is selected from Matrigel ^TM 、Geltrex ^TM And ECM ^TM 。

In one or more embodiments, the medium for preparing the original state PSC/ICLC includes:

(A) DZNep at a final concentration of 5-15nM or CPI-1205 at a final concentration of 0.5-2mM, TSA at a final concentration of 3-30nM or VPA at a final concentration of 0.25-2mM or NaB at a final concentration of 0.25-2mM, preferably TSA at a final concentration of 3-10nM or VPA at a final concentration of 0.25-1mM or NaB at a final concentration of 0.25-1 mM; or DZNep at a final concentration of 5-80nM, preferably 5-50nM, or CPI-1205 at a final concentration of 0.5-5mM, preferably 0.5-3mM, and TSA at 3-10nM, or VPA at a final concentration of 0.25-0.5mM, or NaB at a final concentration of 0.25-0.5 mM;

(B) L-ascorbic acid with a final concentration of 40-70 mug/mL;

(C) LIF with a final concentration of 10-30 ng/mL;

(D) PD0325901 with a final concentration of 0.5-1.5 mu M;

(E) IWR1 or XAV939 at a final concentration of 3-6. Mu.M;

the medium further comprises:

(1) ACTIVIN a or NODAL at a final concentration of 10-25 ng/mL; y27632, thiazovivin or hydroxyfasudil at a final concentration of 0.5-2. Mu.M; and an extracellular matrix in an amount of 0.1% to 0.5% (v/v); or (b)

(2) ACTIVIN a or NODAL at a final concentration of 10-25 ng/mL; and a final concentration of 0.5-2 μm Y27632, thiazovivin or hydroxyfasudil; or (b)

(3) ACTIVIN a or NODAL at a final concentration of 10-25 ng/mL; and an extracellular matrix in an amount of 0.1% to 0.5% (v/v); or (b)

(4) Y27632, thiazovivin or hydroxyfasudil at a final concentration of 0.5-2. Mu.M; and an extracellular matrix in an amount of 0.1% to 0.5% (v/v); or (b)

(5) ACTIVIN a or NODAL at a final concentration of 10-25 ng/mL; or Y27632, thiazovivin or hydroxyfasudil at a final concentration of 0.5-2. Mu.M; or extracellular matrix in an amount of 0.1% -0.5% (v/v).

In one or more embodiments, the medium used to prepare the original PSC/ICLC comprises 10nM DZNep or 1mM CPI-1205;5nM TSA, or 0.5mM VPA, or 0.5mM NaB;50 μg/mL L-ascorbic acid; 20ng/mL LIF;1 μM PD0325901; and 5 μM IWR1 or 5 μM XAV939; and further add: (1) 20ng/mL ACTIVIN A or NODAL, 1. Mu.M Y27632, thiazovivin or hydroxyfasudil, and 0.2% (v/v) extracellular matrix; or (2) 20ng/mL ACTIVIN A or NODAL, and 1. Mu.M Y27632, thiazovivin or hydroxyfasudil; (3) 20ng/mL ACTIVIN A or NODAL, and 0.2% (v/v) extracellular matrix; or (4) 1. Mu.M Y27632, thiazovivin or hydroxyfasudil, and 0.2% (v/v) extracellular matrix; or (5) 20ng/mL ACTIVIN A or NODAL, or 1. Mu.M Y27632, thiazovivin or hydroxyfasudil, or 0.2% (v/v) extracellular matrix.

In one or more embodiments, the medium used to prepare 8CLC comprises DZNep at a final concentration of 40-70nM or CPI-1205 at a final concentration of 2-4 mM; TSA at a final concentration of 10-30nM, or VPA at a final concentration of 0.5-1.5mM or NaB at a final concentration of 0.5-1.5 mM; l-ascorbic acid with a final concentration of 40-70 mug/mL; LIF with a final concentration of 10-30 ng/mL; PD0325901 with a final concentration of 0.5-1.5 mu M; and IWR1 or XAV939 at a final concentration of 3-6. Mu.M, respectively; and further add:

In one or more embodiments, the medium used to prepare 8CLC comprises 50nM DZNep or 3mM CPI-1205;20nM TSA, or 1mM VPA, or 1mM NaB;50 μg/mL L-ascorbic acid; 20ng/mL LIF;1 μM PD0325901; and 5 μM IWR1 or 5 μM XAV939; and further add: (1) 20ng/mL ACTIVIN A or NODAL,1 μ M Y27632, thiazovivin or hydroxyfasudil, and 0.2% (v/v) extracellular matrix; or (2) 20ng/mL ACTIVIN A or NODAL, and 1 μ M Y27632, thiazovivin or hydroxyfasudil; (3) 20ng/mL ACTIVIN A or NODAL, and 0.2% (v/v) extracellular matrix; or (4) 1 μ M Y27632, thiazovivin or hydroxyfasudil, and 0.2% (v/v) extracellular matrix; or (5) 20ng/mL ACTIVIN A or NODAL, or 1 μ M Y27632, thiazovivin or hydroxyfasudil, or 0.2% (v/v) extracellular matrix.

In one or more embodiments, the basal medium used to prepare the original PSC/ICLC and 8CLC medium is selected from one or more of Dulbecco's Modified Eagle Medium (DMEM), minimum Essential Medium (MEM), basal Medium Eagle (BME), RPMI1640, F10, F12, alpha minimum essential Medium (alpha MEM), glasgow Minimum Essential Medium (GMEM), iscove's modified Dulbecco's Medium, neural basal Medium, advanced DMEM/F12; of these, the basal medium is preferably a mixture of higher DMEM/F12 and neural basal medium in a ratio of 1:1 (v/v).

In one or more embodiments, the medium further has added thereto one or more selected from the group consisting of serum substitutes, substituted carbon sources, non-essential amino acids, L-glutamine or substitutes thereof, and antibiotics.

In one or more embodiments, the serum replacement is selected from one or more of KOSR, N2, and B27; preferably, the serum replacement is a mixture of N2 and B27 in a ratio of 1:1 (w/w).

In one or more embodiments, the alternative carbon source is pyruvic acid, such as sodium pyruvate.

In one or more embodiments, the L-glutamine or a substitute thereof is Glutamax containing L-alanyl-L-glutamine dipeptide in 0.85% NaCl ^TM And (3) supplementing.

In one or more embodiments, the antibiotic is selected from penicillin, streptomycin, or a mixture of penicillin and streptomycin.

In one or more embodiments, the method of preparing a primary state PSC/ICLC includes the steps of:

(a) Genetic engineering primate PSCs to reduce the activity of SAH, PRC and/or EZH2 of the PSC by knocking down and/or knocking out one or more associated genes in the cell; and

(b) Culturing the genetically engineered cell obtained in step (a) in a medium comprising: TSA at a final concentration of 3-30nM, or VPA at a final concentration of 0.25-2mM, or NaB at a final concentration of 0.25-2 mM; preferably, TSA is present at a final concentration of 3-10nM, or VPA is present at a final concentration of 0.25-1mM, or NaB is present at a final concentration of 0.25-1 mM; and optionally, DZNep at a final concentration of 5-15nM or CPI-1205 at a final concentration of 0.5-2mM, or TSA at a final concentration of 3-10nM, or VPA at a final concentration of 0.25-0.5mM, or NaB at a final concentration of 0.25-0.5mM and optionally DZNep at a final concentration of 5-80nM, preferably 5-50nM, or CPI-1205 at a final concentration of 0.5-5 mM; l-ascorbic acid with a final concentration of 40-70 mug/mL; LIF with a final concentration of 10-30 ng/mL; PD0325901 with a final concentration of 0.5-1.5 mu M; IWR1 or XAV939 at a final concentration of 3-6. Mu.M; wherein, the culture medium is further added with:

(5) ACTIVIN a or NODAL at a final concentration of 10-25 ng/mL; or Y27632, thiazovivin or hydroxyfasudil at a final concentration of 0.5-2. Mu.M; or an extracellular matrix in an amount of 0.1% to 0.5% (v/v);

preferably, the medium contains: 5nM TSA, or 0.5mM VPA, or 0.5mM NaB;50 μg/mL L-ascorbic acid; 20ng/mL LIF;1 μM PD0325901;5 μM IWR1 or 5 μM XAV939; and optionally 10nM DZNep or 1mM CPI-1205; and wherein the medium is further supplemented with (1) 20ng/mL ACTIVIN a or NODAL,1 μ M Y27632, thiazovivin or hydroxyfasudil, and 0.2% (v/v) extracellular matrix; or (2) 20ng/mL ACTIVIN A or NODAL, and 1 μ M Y27632, thiazovivin or hydroxyfasudil; (3) 20ng/mL ACTIVIN A or NODAL, and 0.2% (v/v) extracellular matrix; or (4) 1 μ M Y27632, thiazovivin or hydroxyfasudil, and 0.2% (v/v) extracellular matrix; or (5) 20ng/mL ACTIVIN A or NODAL, or 1 μ M Y27632, thiazovivin or hydroxyfasudil, or 0.2% (v/v) extracellular matrix.

In one or more embodiments, the steps of the method of preparing 8CLC include:

(a) Genetic engineering primate PSCs or primary PSC/ICLCs to reduce the activity of SAH, PRC and/or EZH2 of PSC or primary PSC/ICLC by knocking down and/or knocking out one or more associated genes in the cell;

(b) Culturing the genetically engineered cell obtained in step (a) in a medium comprising: TSA at a final concentration of 10-30nM, or MVPA at a final concentration of 0.5-1.5m or NaB at a final concentration of 0.5-1.5 mM; l-ascorbic acid with a final concentration of 40-70 mug/mL; LIF with a final concentration of 10-30 ng/mL; PD0325901 with a final concentration of 0.5-1.5 mu M; IWR1 or XAV939 at a final concentration of 3-6. Mu.M; and optionally, a final concentration of 40-70nM DZNep or a final concentration of 2-4mM CPI-1205; and wherein the medium is further supplemented with:

preferably, the medium contains: 20nM TSA, or 1mM VPA, or 1mM NaB;50 μg/mL L-ascorbic acid; 20ng/mL LIF;1 μM PD0325901;5 μM IWR1 or 5 μM XAV939; and optionally 50nM DZNep or 3mM CPI-1205; and wherein the medium is further supplemented with (1) 20ng/mL ACTIVIN a or NODAL,1 μ M Y27632, thiazovivin or hydroxyfasudil, and 0.2% (v/v) extracellular matrix; or (2) 20ng/mL ACTIVIN A or NODAL, and 1 μ M Y27632, thiazovivin or hydroxyfasudil; (3) 20ng/mL ACTIVIN A or NODAL, and 0.2% (v/v) extracellular matrix; or (4) 1 μ M Y27632, thiazovivin or hydroxyfasudil, and 0.2% (v/v) extracellular matrix; or (5) 20ng/mL ACTIVIN A or NODAL, or 1 μ M Y27632, thiazovivin or hydroxyfasudil, or 0.2% (v/v) extracellular matrix.

In one or more embodiments, the primate PSC is selected from the group consisting of:

(i) Cells from the ESC line and/or the ECC line;

(ii) Cells of the iPSC line;

(iii) Cells of ICM of pre-implantation blastocysts cultured in vitro;

(iv) Cells of the ICM of the blastocyst after implantation cultured in vitro;

(v) Cells of embryos from 8-cell stage to morula stage cultured in vitro.

In one or more embodiments, the primate PSC or primary PSC/ICLC can be cultured under one or more conditions selected from the group consisting of: (i) on feeder cells; (ii) on an extracellular matrix without a feeder layer; (iii) in suspension without feeder cells; (iv) under hypoxic or normoxic conditions at about 37 ℃; (v) Passaging every 3 to 4 days with single cells at a split ratio of 1:4 to 1:8; (vi) daily medium changes.

In one or more embodiments, the method further comprises the step of culturing the somatic cells in the presence of a SAH/PRC/EZH2 inhibitor, an HDAC inhibitor, and a WNT/β -catenin signaling inhibitor to reprogram the somatic cells to produce primate primordial state PSC/ICLC.

The fourth aspect of the invention also provides a teratoma produced by the method described in any of the embodiments herein, and cells isolated from the teratoma.

The fifth aspect of the invention also provides an organoid produced by the method described in any of the embodiments herein, and cells isolated from the organoid.

The sixth aspect of the invention also provides an embryoid body produced by the method described in any of the embodiments herein, and cells isolated from the embryoid body.

Drawings

Fig. 1: (a) Schematic of the process of inducing primary PSC/ICLC and 8CLC from primary (prime) PSC with a combination of 4CL,4CL and e4CL media (stepwise e4 CL) or e4CL alone (direct e4 CL). D, day.

(b) KLF17 and TPRX1 immunofluorescence stained images of the original H9 ESC were either untreated or transformed with 4CL (12 days) or e4CL (5 days). Scale bar, 20 μm.

(c) In NHSM,Heat maps of human primary PSC and ICM (cell-like mass) enriched gene expression prior to embryo implantation cultured in 5iLAF, 4 CL. Data were generated using H9 ESC.

(d) In NHSM,Thermal map of totipotent gene expression in 5iLAF or stepwise e4CL (day 5), EPSC and human 8C-embryonic cell cultures in pristine ESCs. Data were generated using H9 ESC.

(e) RT-qPCR validation of the holoenergy in H9 ESCs grown in direct e4CL (day 7). Data are mean ± SEM of fold change compared to the original ESC. n=3 biological replicates. P values were calculated using a two-tailed unpaired student t-test, P <0.001.

Fig. 2: (a) Comparison of return development from human E7 to E3 embryo stage was UMAP map of progressive or direct E4 CL-induced scRNA-seq time course, respectively. Data were generated using H9 ESC.

(b) Step e4CL-day 5 cell RNA-Seq data UMAP visualization, showing 7 clusters, cluster 5 (8 CLC) accounting for 11.9% of total cell number.

(c) Representative frequency and average expression of pluripotency and totipotency genes at early stages of human embryo, and bubble patterns of ESCs in the original state that were not treated or transformed with 4CL (day 8 [ 2 nd generation ] and day 12 [ 3 rd generation ]) and e4CL (day 5C 5[8CLC ] and non-8 CLC [ all other cluster additions ]). D, day.

(d) Violin plots with representative log normalized expression of early human embryo-enriched TE at early stage of human embryo and 10 th generation of human ESC compared to original ESC, 4CL-day 12 original ESC and 8 CLC.

Fig. 3: (a) G-banding pattern representative images of the originating H9 and originating iPSC-4 were cultured in 4CL for 15 generations. Each figure counts 20 cells at metaphase.

(b) G-banding pattern representative images of originating H9 and iPSC-4 cultured in step e4CL (day 5). Each figure counts 20 cells at metaphase.

Fig. 4: (a) In the original state, 4CL (day 12), 5iLAF,Violin plots of global CpG methylation levels were detected by RRBS for human PSCs, human 8C embryos and ICM cultured in NHSM, stepwise e4CL (day 5) and direct e4CL (day 7). Generating data with H9 ESC

(b) In the original state, 4CL (day 12), 5iLAF,Heat maps of CpG methylation levels in control areas of post-implantation embryo imprinting for human PSCs and ICMs cultured in NHSM and step e4CL (day 5).

(c) Under conditions of origin, 4CL (day 12), 5iLAF,Genomic map traces of PSCs cultured under NHSM, stepwise e4CL (day 5) and direct e4CL (day 7), human 8C embryos and ICM, indicating CpG methylation levels at the original state pluripotent (blue) and totipotent (red) sites. Each bar represents a single CpG, and the heights represent the percentage of methylation.

Fig. 5: (a) UMAP gene scores for all genes in scaTAC-seq were visualized, with the original ESCs untreated (red), 4CL (day 12; blue) or step e4CL (day 5; green).

(b) And (c) highlighting the originating state (ZIC 2) based on figure a, sharing gene score UMAP visualization of the original state pluripotency/8 CLC (DPPA 3) and totipotency (ZSCAN 5B, ZNF280A, ARGFX) genes projected onto each cell in the originating state ESC-seq untreated or transformed by 4CL (day 12) and step e4CL (day 5).

(d) Genome browse of original multipotent KLF17 and totipotent ZSCAN4 sites showing chromatin accessibility, H3K27ac levels and transcription factor DNA binding motif positions by tracking.

Fig. 6: (a) upper graph: schematic of the insertion of EGFP into the TPRX1 site (for chimeric experiments), and donor constructs for generating TPRX1-EGFP reporter cell lines. The following figures: TPRX1-EGFP knocked in cells and immunostained with GFP as a result by e4CL step culture (day 5) and anti-TPRX1 ⁺ Signal agreement (left panel). Ruler: 10 μm. GFP from TPRX1-EGFP cells in step e4CL (day 5) ⁺ Cell percentage FACS analysis graph (right panel).

(b) Representative pluripotency and totipotency genes expression frequency and average expression bubble patterns for day 12, 4CL virgin ESC and sorted 8CLC, human early embryo stage and 10 th generation of human ESC compared to virgin ESC.

Fig. 7: expression level histogram of ICM and primer markers in H9, H1, HUES1 and WIBR3 human ESC lines, which had been transformed to original PSC/ICLC using 4CL Medium 1.

Fig. 8: in 4 CL-containing culture medium 1, geltrex ^TM RT-qPCR data histogram of significant induction of ICM marker genes KLF17, DNMT3L, DPPA, STELLA, TFCP2L1, KLF4, MAEL and REX1 before implantation in ICLC transformed on coated dishes.

Fig. 9: in the original state PSC/ICLC transformed with 4CL Medium 1 suspension, pre-implantation ICM marker genes KLF17, DNMT3L, DPPA5, STELLA, TFCP2L1, KLF4, MAEL and REX1 were significantly induced by RT-qPCR data histograms. In the bar graph, each gene in the left column represents cells cultured on feeder cells, and the right column represents cells cultured in suspension.

Fig. 10: RT-qPCR data histogram of significant induction of pre-implantation ICM marker genes KLF17, DNMT3L, DPPA5, STELLA, TFCP2L1, KLF4, MAEL and REX1 using 4CL Medium 2 (A), 4CL Medium 3 (B), 4CL Medium 4 (C) transformed original PSC/ICLC, respectively.

Fig. 11: (A) schematic diagrams of two methods of generating 8 CLC. Briefly, an original human PSC medium (e.g., mTeSR 1) was replaced with e4CL medium or 4CL medium. Cells were then either continuously cultured in e4CL or replaced with e4CL after two passages in 4CL medium. (B) Expression level histogram of selected original multifunctional marker genes in H9-originated cells and H9-e4CL cells. (C) Bar graph of expression levels of selected original multifunctional marker genes in H9-e4CL cells and H9-4CL cells. (D) The level of 8C-specific gene expression induced by both methods was similar. (E) Immunofluorescence microscopy imaging of ZSCAN4 (green) or DAPI nuclear counterstain (blue) expressed in the original states H9, H9-4CL and H9-e 4CL.

Fig. 12: in 8CLC suspension transformed using e4CL medium, 8C marker genes ZSCAN4, ARGFX, TPRX1, ZNF280A and ZSCAN5B were significantly induced by RT-qPCR data histograms. In the bar graph, the left column of each gene represents cells cultured on feeder cells, and the right column represents cells cultured in suspension.

Fig. 13: bar graphs of RT-qPCR data for significant induction of 8C marker genes ZSCAN4, ARGFX, TPRX1, ZNF280A, ZSCAN5B, DUXA, DUXB and MBD3L2 from 8CLC transformed from various hPSC lines. The figure shows that these genes are expressed in the original HN10 and UH10 in very low amounts.

Fig. 14: (a) Representative images of teratomas from originating ESCs or from 4CL (15 th generation) and sorted 8 CLCs. H9 ESCs were used for this experiment.

(b) Teratoma hematoxylin and eosin staining patterns generated from 4CL (15 th generation) and 8CLC after sorting. Tissue representative images corresponding to the three germ layers are shown. Scale bar: 50 μm. H9 ESCs were used for this experiment.

(c) UMAP visualization of identified cell types in 8CLC, e4CL-day 5 cells, 4CL naive ESCs and originating ESC teratoma scRNA-seq from sorting. H9 ESCs were used to generate these teratoma cell types.

Fig. 15: UMAP visualization of identified cell types in scRNA-seq generated from sorting 8CLC, step e4CL-day-5 cells, 4CL original ESCs and original ESCs, respectively, is highlighted based on FIG. 14 c.

Fig. 16: (a) Histogram of the ratio distribution of different teratomas to identified cell types.

(b) Relative proportion bar graph of different teratomas for each germ layer (ectodermal, mesodermal and endodermal) and extraembryonic (trophoblast) lineages.

Fig. 17: (a) Trophectoderm cell types UMAP visualization identified from scRNA-seq of teratomas derived from sorting 8CLC, e4CL-day-5 cells, 4CL naive ESCs and originating ESCs. All types of teratoma cells were cultured using H9 ESC.

(b) Bubble pattern of frequency and average expression level of marker genes in cell subtypes of the extraembryonic trophoblast lineage.

(c) Sorting 8CLC, e4CL-day-5 cells, 4CL naive ESCs, and originating ESCs produced a histogram of the relative proportions of teratomas versus the extracellular trophoblast lineage cell subtype.

(d) UMAP visualization of the distribution and expression of relevant markers in the indicated teratoma trophoblast cells is shown according to FIG. 14 c.

Fig. 18: (a) UMAP visualization of labeled cell subsets of the original PSC, 4CL naive ESC, stepwise e4CL-day-5 cells and sorted 8 CLC-derived teratoma immune cells.

(b) Bubble pattern of frequency and average expression level of immunomarker genes in different immune cell subtypes.

(c) Distribution histogram of 8CLC, stepwise e4CL-day-5, 4 CL-primary and primary PSC-derived teratomas against different immune cell subtypes after sorting.

Fig. 19: (a) Distribution UMAP visualization of teratoma cells was obtained from the original PSC, 4CL original PSC, e4CL cells and sorted 8 CLC.

(b) Annotated subtype UMAP visualization of neuronal cells from originating PSC, 4CL primary PSC, e4CL cells and sorted 8CLC derived malformations.

(c) Histogram of the relative contributions of different neuronal cell types to the indicated teratomas.

(d) Contribution bar graph of different teratomas to the identification of cell types.

Fig. 20: (a) Brain organoid cell distribution UMAP visualization derived from original state and 4CL original state PSCs.

(b) Annotated subtype UMAP visualization of neural cells derived from brain organoids from both the original state and 4CL original state PSC.

(c) Bar graph of the ratio of individual cell types in two brain organoids.

(d) Bar graph of the proportion of two brain organoids in each cell type.

Fig. 21: (a) Distribution UMAP visualization of EB (embryoid body) derived from PSC in the original state and 4CL in the original state.

(b) Annotated cell type UMAP visualization in PSC derived EBs from the original state and 4CL original state.

(c) Histogram of the duty cycle of different cell types in a given EB.

(d) Histogram of the duty cycle of different EBs in a given cell type.

Detailed Description

Current methods of generating and maintaining human PSC in raw form (Chan, goke et al, 2013; takashima, guo et al, 2014; theunissen, powell et al, 2014) and exhibit similar characteristics to human pre-implant ICM. The original human PSC generated using existing methods is a problem that remains to be solved, such as long time induction times, varying levels of original specific gene expression, transgene dependency, genome instability and loss of imprinting, low capacity for multiple lineage differentiation, and lack of heterologous chimerism. None of these studies reported the generation of cells near stage 8C. Furthermore, there is no report describing the generation of embryonic external lineages in teratomas generated using human primary or originating PSCs, nor does the totipotent of the primary PSC differentiation in vivo expand to be explored compared to the original PSC without transformation.

To overcome the above problems, the inventors first screened a panel of inhibitors against epigenetic regulators and various signaling pathways associated with the development of the pre-implantation inner cell mass in humans, and found that three basic modulators (JAK/STAT 3 activator, MAPK/ERK inhibitor and tankyrase inhibitor) could activate a molecular network that controls the pre-implantation inner cell mass-like state of human PSCs. The inventors have also found that SAH/PRC/EZH2 inhibitors and HDAC inhibitors bring the epigenetic and transcriptomic status of the cultured cells closer to the human pre-implantation inner cell mass, they convert the original PSC into the original PSC, which has all the main characteristics of the human pre-implantation inner cell mass as described in the background section. Notably, the inventors have also found that activation of the WNT/β -catenin signaling pathway inhibits the transition from the original state PSC to the original state PSC/ICLC. Therefore, modulators such as CHIR99021, a GSK inhibitor which activates WNT/β -catenin signals (a WNT/β -catenin signal pathway activator widely used in the published state and expanding the culture conditions of PSCs), should be removed from the culture medium, and modulators such as IWR1, XAV939 which inhibit WNT/β -catenin signal pathways are necessary. Thus, in preferred embodiments of various aspects of the application, including the media, kits, compositions and methods described herein, CHIR99021, GSK inhibitor, and any agent that activates WNT/β -catenin signaling pathway are not included in the media, kits or compositions, and are not used in methods of culturing cells.

Thus, the present application provides a variety of methods and chemically defined media to promote stable primate PSC/ICLC production. The methods described herein are applicable to many human and non-human primate PSC lines that are validated in their original state by surface markers such as SSEA-3, SSEA-4, TRA-1-81, and TRA-1-60, or in their pre-implantation ICM-like state as validated by expression of genes such as DNMT3L, STELLA, DPPA5 and KLF 17. Primate PSC lines useful in the present application include, but are not limited to, traditional primate PSCs and ICM-like PSCs. The methods described herein can also be used to isolate primary PSC/ICLC from primate pre-implantation inner cell mass. The method does not require a transgene and primate PSC can be converted to original PSC/ICLC in about 2 weeks under one culture condition.

To our knowledge, there is currently no method for in vitro induction of primate 8CLC. To achieve this, the inventors have further optimized the formulation for inducing ICLC and found that only increasing the doses of SAH/PRC/EZH2 inhibitor and HDAC inhibitor in the medium, resulted in the conversion of the original human PSC and/or ICLC to 8CLC. Thus, the present application provides a chemically defined medium that promotes the production of primate 8-cell embryoid-like cells (8 CLC). The methods described herein are applicable to many human and non-human primate PSC lines that are in an originating state as verified by expression of pluripotent surface marker genes such as SSEA-3, SSEA-4, TRA-1-81, and TRA-1-60, or in a pre-implantation ICM-like state as verified by expression of genes such as DNMT3L, STELLA, DPPA and KLF 17. Primate PSC lines useful in the present application include, but are not limited to, originating primate PSCs and ICM-like PSCs. The methods described herein can also be used to isolate 8CLC from primate 8-cell embryos. The method does not require a transgene and converts to 8CLC in about 1 week under one culture condition. In fact, activation of the WNT/β -catenin signaling pathway also inhibited 8CLC formation. Therefore, modulators such as CHIR99021, a GSK inhibitor which activates the WNT/β -catenin signaling pathway, which is a WNT/β -catenin signaling pathway activator widely used in the published state and expanding PSC culture conditions, should be excluded from the culture conditions, and modulators such as IWR1 or XAV939 which inhibit the WNT/β -catenin signaling pathway are necessary.

ICLC/primary PSC and 8CLC are able to capture features of their corresponding embryo development stages in vivo and have fewer epigenetic abnormalities. However, the differentiation schemes of ICLC/primary PSC and 8CLC are still lacking. Thus, we used these cells for various in vivo and in vitro differentiation to form teratomas, brain organoids and EB.

Teratomas produced by ICLC/primary PSC and/or 8CLC contained a high percentage of HSCs, LPCs and a significant percentage of extra-embryonic cells compared to teratomas produced by primary PSCs. By reconstructing the differentiation trajectories of HSCs and LPCs, we demonstrate that HSCs and LPCs are able to differentiate into functional cell types of hematopoietic, hepatic and embryonic lineages, respectively. Brain organoids generated from ICLC/primary PSC include a higher percentage of neuroepithelial cells and retinal progenitor cells, which can produce different types of neuronal cells. A higher percentage of progenitor cells may lead to higher complexity and more functional brain organoids. In addition, EBs produced from ICLC/primary PSC contain more neuroepithelial cells, trophoblast cells and endodermal epithelial cells and can be a good source of derived human cells or tissues. In summary, we have developed a method for producing primate tissues, cells and biomolecules with therapeutic potential.

The details of the present application will be described below. It should be understood that the features described in the various embodiments may be combined with each other to form preferred solutions, which solutions are also within the scope of the application.

I. Terminology

Unless otherwise defined, all terms used herein have the meanings commonly understood by those skilled in the art. In order to facilitate an understanding of the present application, some terms used herein are defined as follows.

As used in the specification and the claims, the singular forms "a", "an" and "the" include plural referents unless the context clearly dictates otherwise. For example, the term "cell" includes a plurality of cells, including mixtures thereof.

All numerical indicators such as pH, temperature, time, concentration and molecular weight (including ranges) are approximations. It is to be understood that all numerical designations are preceded by the term "about", although not always explicitly stated. It is also to be understood that the agents described herein are merely examples, although not always explicitly described, and equivalents thereof are known in the art.

The term "basal medium" as used herein refers to any medium capable of supporting cell growth. Basal media provides standard inorganic salts such as zinc, iron, magnesium, calcium and potassium, as well as vitamins, glucose, buffer systems and key amino acids. Minimal media that may be used in the present application include, but are not limited to, dulbecco's Modified Eagle Medium (DMEM), minimal Essential Medium (MEM), basal Medium Eagle (BME), RPMI1640, F10, F12, alpha minimal essential Medium (alpha MEM), glasgow Minimal Essential Medium (GMEM), iscove's modified Dulbecco's Medium, neural basal Medium, and DMEM/F12. The person skilled in the art knows how to select a basal medium suitable for the cells to be cultivated. In a preferred embodiment, the basal medium used in the present application is a 1:1 (w/w) mixture of DMEM/F12 and neural basal medium.

The term "serum-free" refers to the absence of any serum of any species, including, but not limited to, the absence of fetal bovine serum, calf serum, human serum, and the like, or a combination thereof.

The term "serum replacement" as used herein refers to an additive in a basal medium that is used to partially or completely replace serum to support cell survival and growth. Serum substitutes generally include insulin, metalloproteins, trace elements, vitamins, and the like. These factors are not normally contained in the basal medium but are provided by serum which is commonly used to culture cells. The serum replacement comprises at least one or more of the following components that support cell growth: one or more insulin and insulin substitutes, one or more metalloproteins and metalloprotein substitutes, one or more trace elements, one or more vitamins, one or more amino acids, one or more hormones and hormonal compounds, serum albumin or serum albumin substitutes, and one or more lipids, etc. Various commercial serum substitutes are known in the art, including KOSR, N2, B27, insulin transferrin selenium supplement (ITS), G5, and the like, readily available to those skilled in the art. These alternatives are of well-defined composition, and therefore the concentration of each component can be determined according to their respective proportions in the medium.

The skilled person can readily configure the serum replacement according to the prior art, the type of cells to be cultured, etc. Preferably, the serum replacement used herein is a mixed additive obtained by mixing KOSR, N2 and/or B27 in a certain ratio. More preferably, the serum replacement used herein is a 1:1 (w/w) mixture of N2 and B27.

The term "Primate" or "Primate" as used herein refers to animals belonging to the order primates (primates). Primates include humans and non-human primates. Non-human primates include animals of the order Simian (Prosimian) and Simian (Simiae). Specific non-human primates include, but are not limited to, macaques, lemurs, gibbons, gorillas and baboons.

As used herein, "pluripotent stem cells" (PSCs) refer to iPSCs produced by reprogramming pluripotent cells and somatic cells obtained from an embryo at any time prior to gastrulation. Depending on its source and method of cultivation, PSCs may be in different states, including originating PSCs, original PSCs, expanded PSCs, and expanded PSCs (Gafni et al, 2013; gao et al, 2019; takashima et al, 2014; theunissen et al, 2014; yang et al, 2017). PSC is characterized by the ability to produce offspring of different cell types under appropriate conditions, which are derivatives of three germ layers (endodermal, mesodermal and ectodermal). These can be determined according to technical tests standard in the art, for example 6 to 12 week old SCID mice' ability to form teratomas, and can also produce different cell types of placenta under appropriate conditions. PSC cultures are described as "undifferentiated" when a substantial proportion of stem cells and their derivatives in a population exhibit morphological characteristics of undifferentiated cells, thus distinguishing them from differentiated cells of embryonic or adult origin. It is understood that colonies of undifferentiated cells within the population may be surrounded by adjacent differentiated cells.

Various types of stem cells may be used in the present application. Particularly suitable for use in the present application are primate pluripotent stem cells. Non-limiting examples are primary cultures or established lines of ESCs and iPSCs. Any non-primate pluripotent stem cells may also be used in the present application.

In one or more embodiments, primate PSCs useful in the present application can be selected from the group consisting of:

(i) Cells from the ESC line and/or the ECC line;

(ii) Cells of the iPSC line;

(iii) Cells of ICM of pre-implantation blastocysts cultured in vitro;

(iv) Cells of the ICM of the blastocyst after implantation cultured in vitro; and

(v) Cells of embryos from 8-cell stage to morula stage cultured in vitro.

Non-limiting PSCs include, but are not limited to, any established cell line in the art, such as human ESC lines, e.g., H1 (male), H9 (female), HN10 (female), HUES1 (female), and WIBR3 (female); human iPSC lines, such as CBC14 (female), C11 (female), phoenix (female), diPS 1016SevA (male), STiPS O-XX1 (female), and UH10 (male).

As used herein, the term "teratoma" is a model of multiple lineage development that studies human and other animal development, comprising various cell types of the three germ layers. Teratomas can be used to analyze the effects of genetic perturbation of multiple cell types simultaneously, and can be molecularly sculpted by microRNA (miRNA) regulated suicide gene expression to enrich specific tissues. Teratomas can be used as a platform for modeling multiple lineage development, genetic screening of pan-tissue function, and tissue engineering.

As used herein, the term "organoids" refers to 3D "micro-organs" derived from an original state PSC, original state PSC/ICLC or 8CLC, in which cells spontaneously assemble into functional cell types that mimic the differentiation of their in vivo counterparts in structure and function.

As used herein, the term "embryoid body" (EB) refers to a pluripotent cell aggregate induced to differentiate by a combination of changing the medium (removing factors that support pluripotency) and allowing the cells to interact in a 3D structure. EB forms a 3D structure under differentiation conditions, typically used as an initiation process for directional differentiation. EB contains different germ layer cell types and can be used as a model for rapid generation of target cell types in vitro.

II culture medium

The media disclosed herein are chemically defined media that can effectively transform primate PSCs from an original state to a pre-implantation inner cell mass-like state, thereby producing pre-implantation inner cell mass-like original state PSC/ICLC within 2 weeks without the need to pick colonies. The culture medium of the present application can also be used to generate 8-cell embryoid-like cells (8 CLC) based on the transformation of primate PSCs from an originating state and/or a pre-implantation inner cell mass-like state to an 8-cell embryoid-like state within about 1 week. Thus, such a medium may also be referred to as a "transformation medium" in the present application. In some embodiments, the culture medium of the application may also support the production, passage, and/or survival, self-renewal, and proliferation of cells following resuscitation of cells in a cell mass-like state within the pre-implantation. In some other embodiments, the culture medium of the application may also support the passage of cells in a pre-implantation endo-cell mass-like state on extracellular matrix and/or survival, self-renewal and proliferation after resuscitation without the need for feeder cells or conditioned medium. In some embodiments, the culture medium of the application may also support passage of cells in suspension in a cell mass-like state prior to implantation and/or survival, self-renewal and proliferation after resuscitation without the need for feeder cells or conditioned medium. In some other embodiments, the culture medium of the application may also support passage of cells in a cell mass-like state on feeder cells prior to implantation and/or survival, self-renewal and proliferation after resuscitation. Preferably, the chemically defined medium of the application is a serum-free medium.

The culture medium of the application comprises a basal medium and is supplemented with PRC and/or EZH2 inhibitors, HDAC inhibitors and WNT/beta-catenin signaling pathway inhibitors, and optionally one or more components selected from L-ascorbic acid, JAK/STAT3 signaling activators and MAPK/ERK signaling inhibitors. The basal medium is capable of supporting cell growth, and in particular, is capable of supporting the growth of PSCs in humans and non-human primates. Preferably, the basal medium used in the present application is a 1:1 (v/v) mixture of higher DMEM/F12 and neural basal medium. It is understood that inhibitors of SAH can also achieve the effect of inhibiting PRC and EZH 2. Thus, in some embodiments, the PRC and/or EZH2 inhibitor is an SAH inhibitor. In the present application, the term "SAH/PRC/EZH2 inhibitor" refers to an inhibitor of SAH, PRC and/or EZH 2.

Under these culture conditions, the presence of SAH/PRC/EZH2 inhibitors is critical for the induction of a variety of mediators (including STELLA, DNMT3L and MAEL) that control the original state molecular network in humans. STELLA is a DNA methylation regulator. Its ectopic overexpression in somatic cells can induce global demethylation of DNA by interfering with the function of the DNA methylation regulator UHRF 1. UHRF1 dysfunction caused by STELLA deficiency can lead to the accumulation of abnormal DNA methylation during ovum development (Li et al, 2018). Induction of STELLA was dose dependent. The inventors further revealed a functional role for STELLA, and found that STELLA knockout prevented the induction of primary PSC/ICLC and 8 CLC. During the conversion of the original PSC to the original PSC/ICLC, STELLA deletion caused the failure of the pre-implantation ICM markers, including KLF17, DPPA5, DNMT3L, TFCP L1 and MAEL, to be induced. In the course of transformation of the original PSC and ICLC into 8CLC, the 8C markers including TPRX1, TRIM60, KHDC1L, YPEL, ALPG, ZNF280F, FAM151A and CCNA1 were not induced in the case of STELLA deletion. As demonstrated by the present application, overall DNA methylation levels of STELLA knockout cells were significantly elevated compared to wild type during transformation with 4CL or e4CL medium. Thus, STELLA is a requirement for controlled demethylation of DNA during the conversion to ICLC and 8 CLC. In summary, the present application found that SAH/PRC/EZH2 inhibitors can promote induction of ICLC and 8CLC by resetting histone modification and DNA methylation status.

Any substance that can be used as an SAH/PRC/EZH2 inhibitor can be used in the culture medium of the present application, including but not limited to DZNep (CAS number: 102052-95-9, SAH inhibitor) and CPI-1205 (CAS number: 1621862-70-1, PRC/EZH2 inhibitor). In the medium of the present application, the SAH/PRC/EZH2 inhibitor may be used alone or in combination, usually in their respective conventional amounts, and in amounts not to cause cell death. For example, the final concentration of DZNep in the medium may be 5 to 80nM, preferably 5 to 50nM, and the final concentration of CPI-1205 may be 0.5 to 5mM, preferably 1 to 3mM. In one or more embodiments, the SAH/PRC/EZH2 inhibitor is a PRC inhibitor.

Any substance that can act as an HDAC inhibitor can be used in the culture medium of the present application, including but not limited to trichostatin a (TSA), valproic acid (VPA), and sodium butyrate (NaB). In the culture medium of the present application, the HDAC inhibitors may be used alone or in combination, typically in their respective conventional amounts, and in amounts which do not result in cell death. For example, the final concentration of TSA in the medium may be 3 to 30nM, preferably 3 to 25nM, the final concentration of VPA may be 0.25 to 2mM, preferably 0.5 to 1.5mM, and the final concentration of NaB may be 0.25 to 2mM, preferably 0.5 to 1.5mM.

The inventors have also found that when both the SAH/PRC/EZH2 inhibitor and the HDAC inhibitor are used in relatively high concentrations, the primary PSC and/or ICLC can be converted to 8CLC by the culture medium of the present application. Specifically, in some embodiments, to produce 8CLC, the DZNep may be 40nM or higher, such as 40-80nM, preferably about 50nM, when each is used alone; CPI-1205 may be 2mM or greater, such as 2-5mM, preferably about 3mM; TSA may be 10nM or higher, such as 10-30nM, preferably about 20nM; VPA may be 1mM or more, such as 1-2mM, preferably about 1.5mM; and NaB may be 1mM or more, such as 1-2mM, preferably about 1.5mM. It will be appreciated that when two or more SAH/PRC/EZH2 inhibitors or two or more HDAC inhibitors are used, the final concentration of each SAH/PRC/EZH2 inhibitor or each HDAC inhibitor should be reduced to an amount sufficient to induce 8CLC by a combination of these SAH/PRC/EZH2 inhibitors or HDAC inhibitors. These amounts can be readily determined by one of ordinary skill in the art based on the disclosure of the present application and conventional knowledge in the art.

Furthermore, it is also understood that excessive SAH/PRC/EZH2 inhibitors and HDAC inhibitors may lead to cell death. Thus, to induce primary PSCs/ICLC while minimizing cell death, one or both of SAH/PRC/EZH2 inhibitors and HDAC inhibitors may be used at lower concentrations. Specifically, when used alone, DZNep may have a final concentration of 5 to 15nM, preferably about 10nM, CPI-1205 may have a final concentration of 0.5 to 3mM, preferably about 1mM, TSA may have a final concentration of 3 to 10nM, preferably 4 to 6nM, more preferably about 5nM, VPA may have a final concentration of 0.25 to 1mM, preferably 0.5mM, and NaB may have a final concentration of 0.25 to 1mM, preferably 0.5mM. In some embodiments, the SAH/PRC/EZH2 inhibitor is used in a relatively high concentration range, such as a final concentration of DZNep of 5 to 80nM, preferably 5 to 50nM, a final concentration of CPI-1205 of 0.5 to 5mM, preferably 1 to 3mM, and the HDAC inhibitor is used in a relatively low concentration range, such as a final concentration of TSA of 3 to 10nM, preferably 4 to 6nM, a final concentration of VPA of 0.25 to 0.5mM, and a final concentration of NaB of 0.25 to 0.5mM. In some embodiments, the SAH/PRC/EZH2 inhibitor is used in a relatively low concentration range, e.g., the final concentration of DZNep may be 5 to 15nM, the final concentration of CPI-1205 may be 0.5 to 2mM, while the HDAC inhibitor is used in a relatively high concentration range, e.g., the final concentration of TSA may be 3 to 30nM, preferably 3 to 25nM, the final concentration of VPA may be 0.25 to 2mM, and the final concentration of NaB may be 0.25 to 2mM. Such media can convert primate PSCs to primary PSCs/ICLCs.

Inhibitors of the WNT/beta-catenin signaling pathway in the culture medium of the application include tankyrase inhibitors that inhibit classical WNT signaling. Any known tankyrase inhibitor may be used, particularly those commonly used in stem cell culture, including, but not limited to IWR1 (CAS No. 1127442-82-3) and XAV939 (CAS No. 284028-89-3). The tankyrase inhibitor may be used in an amount commonly used in culturing stem cells. Exemplary final concentrations of the tankyrase inhibitor may range from 2 to 8 μm, preferably from 3 to 6 μm. For example, for IWR1 and XAV939, their respective final concentrations in the media of the application may be in the range of 2 to 8. Mu.M, preferably 3 to 6. Mu.M, more preferably about 5. Mu.M. The tankyrase inhibitor may be used in combination of two or more, while reducing the amount of each inhibitor used.

L-ascorbic acid was found to improve the production and maintenance of mouse iPSC (similar to mouse ESCs) from somatic cells by enhancing the Jumonji domain-containing histone demethylase as described in application No. CN 200910041331.9, the contents of which are incorporated herein by reference. Thus, the inventors hypothesized that L-ascorbic acid also has a similar effect on the formation of a primate pre-implantation endo-cell mass-like state. Through appropriate testing, the inventors found that L-ascorbic acid could potentially increase the expression level of inner cell mass specific genes (such as DNMT3L, STELLA, DPPA5 and KLF 17) when used at final concentrations of 40 to 70. Mu.g/mL. In a preferred embodiment, L-ascorbic acid is used at a final concentration of about 50 μg/mL.

L-ascorbic acid derivatives, which are similar compounds having similar structure and antioxidant activity to L-ascorbic acid, can also be used in the present application. These derivatives are more stable or more easily absorbed by cells while maintaining the biological activity of L-ascorbic acid. L-ascorbic acid derivatives include, but are not limited to, L-ascorbyl phosphate and L-ascorbyl organic esters, such as L-ascorbyl palmitate. The amount of L-ascorbic acid derivative in the medium is not limited, but should generally be sufficient to produce a sufficient amount of L-ascorbic acid as described above.

The culture medium of the application may contain one or more activators of Janus kinase (JAK)/signal transduction and activator of transcription 3 (STAT 3) (i.e., JAK/STAT 3) signaling that synergistically induce the early embryo-specific gene subsets of the application. Any known JAK/STAT3 activator may be used, particularly, those commonly used in stem cell culture are preferred. One of the JAK/STAT3 activators is LIF. LIF, as used herein, refers to leukemia inhibitory factor, a growth factor that is commonly added to culture stem cells. Preferred LIF is human LIF. The amount of JAK/STAT3 activator is a common amount that can be used in stem cell culture, and exemplary final concentrations can typically be 10 to 50ng/mL. For example, for LIF, especially human LIF, the final concentration in the medium of the present application may be from 10 to 50ng/mL, preferably from 10 to 30ng/mL, more preferably about 20ng/mL.

The culture medium of the application may contain one or more inhibitors of mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK) (i.e., MAPK/ERK) signaling that help reduce DNA methylation in concert with other components in the culture medium of the application. Any known MAPK/ERK inhibitor may be used, particularly those commonly used in stem cell culture are preferred. One such MAPK/ERK inhibitor is PD0325901 (CAS number 391210-10-9). The amount of MAPK/ERK inhibitor is a common amount that can be used in stem cell culture, and exemplary final concentrations can range from 0.5 to 3. Mu.M, preferably from 0.5 to 1.5. Mu.M. For example, for PD0325901, its final concentration in the medium of the present application may be 0.5 to 3. Mu.M, preferably 0.5 to 1.5. Mu.M, more preferably about 1. Mu.M.

In one or more embodiments, the medium comprises DZNep at a final concentration of 5 to 15nM or CPI-1205 at a final concentration of 0.5 to 2 mM; TSA at a final concentration of 3 to 30nM, or VPA at a final concentration of 0.25 to 2mM or NaB at a final concentration of 0.25 to 2 mM; preferably TSA at a final concentration of 3 to 10nM, or VPA at a final concentration of 0.25 to 1mM or NaB at a final concentration of 0.25 to 1 mM; 40-70 ug/mL of L-ascorbic acid; LIF with final concentration of 10-30 ng/mL; PD0325901, the final concentration is 0.5-1.5 mu M; the final concentration of IWR1 or XAV939 is 2 to 8. Mu.M, preferably 3 to 6. Mu.M, respectively; in one or more embodiments, the medium of the present application comprises DZNep at a final concentration of 5 to 80nM (preferably 5 to 50 nM) or CPI-1205 at a final concentration of 0.5 to 5mM (preferably 0.5 to 3 mM); the final concentration of TSA is 3-10M, the final concentration of VPA is 0.25-0.5 mM, and the final concentration of NaB is 0.25-0.5 mM; the final concentration of IWR1 or XAV939 is 2 to 8. Mu.M, preferably 3 to 6. Mu.M, respectively; the final concentration of L-ascorbic acid is 40-70 mug/ml; LIF with final concentration of 10-30 ng/mL; PD0325901 has a final concentration of 0.5 to 1.5 mu MIWR1 or XAV939 of 3 to 6 mu M. More preferably, the medium for this application comprises 10nM DZNep or 1mM CPI-1205;5nM TSA, or 0.5mM VPA, or 0.5mM NaB;50 μg/ml L-ascorbic acid; LIF at 20 ng/mL; 1 μM PD0325901; 5. Mu.M IWR1 or 5. Mu.M XAV939. These media can be used to convert primate PSCs to primary PSCs/ICLCs.

In one or more preferred embodiments, the medium of the present application comprises DZNep at a final concentration of 40 to 70nM or CPI-1205 at a final concentration of 2 to 4 mM; the final concentration of TSA is 10-30 nM, the final concentration of VPA is 0.5-1.5 mM, and the final concentration of NaB is 0.5-1.5 mM; the final concentration of L-ascorbic acid is 40-70 g/ml; LIF with final concentration of 10-30 ng/mL; PD0325901, the final concentration is 0.5-1.5 mu M; the final concentration of IWR1 or XAV939 was 3-6. Mu.M, respectively. The medium for this application more preferably comprises 50nM DZNep or 3mM CPI-1205;20nM TSA, or 1mM VPA, or 1mM NaB;50ug/ml L-ascorbic acid; LIF at 20 ng/mL; 1 μM PD0325901; 5. Mu.M IWR1 or 5. Mu.M XAV939. These media are preferably used to convert primate PSCs or primary PSCs/ICLCs to 8CLCs.

The culture medium used in the present invention may further comprise at least one or more additives from the group consisting of extracellular matrix, ACTIVIN/NODAL signal activator and ROCK inhibitor.

The expression level of NODAL (activator of ACTIVIN/NODAL signal) is increased in primary PSCs/ICLCs and 8CLCs compared to human primary PSCs. This observation suggests that the ACTIVIN/NODAL signal is endogenous/spontaneously activated during the conversion process and during self-renewal. Thus, in some embodiments of the subject application, the medium further comprises an activator of the ACTIVIN/NODAL signal to accelerate the conversion process. Any known ACTIVIN/NODAL signal activator may be added to the culture medium of the present application, including but not limited to human ACTIVIN a and human NODAL, the amino acid sequences of which are well known. In the medium used in the present application, the final concentration is about 10 to 25ng/mL, preferably about 20ng/mL, of human ACTIVIN/NODAL. A combination of human ACTIVIN/NODAL may also be used. In general, the total concentration of human ACTIVIN A and human NODAL in the culture solution is between 10 and 25ng/mL, preferably about 20 ng/mL.

After conversion to primary PSCs/ICLCs and/or 8CLCs, single cell passaging cell survival no longer requires ROCK signal inhibition. However, providing low concentrations of ROCK inhibitors can increase the yield of primary PSCs/ICLCs and 8CLCs, which would be advantageous for scale up of culture. Thus, in some embodiments of the application, the culture medium further comprises a ROCK inhibitor. Any known ROCK inhibitor may be used in the media currently in use, including but not limited to Y27632 (CAS No. 146986-50-7), thiozovivin (CAS No. 1226056-71-8), hydroxyfasudil (CAS No. 105628-72-6). ROCK inhibitors may be used at final concentrations of 0.5 to 2uM, preferably about 1 uM. Two or more ROCK inhibitors may be used in combination, and the total concentration in the medium is 0.5 to 2. Mu.M, preferably about 1. Mu.M.

The present application has found that when PSCs are cultured in the media of the present application, they can be transformed and maintained in suspension culture without feeder cells, and the transformed cells can self-renew and proliferate in the manner of spherical clones. Thus, in some embodiments of the application, the methods, culture conditions, and media are feeder cells-free.

In some other embodiments, the inventors have discovered that providing additional cell matrix during transformation and maintenance will promote spheroid-like cell colonies. Under such conditions, more than 90% of PSCs can be transformed into hemispherical colonies during transformation Internal cell mass markers such as DNMT3L and KLF17 are expressed. Thus, in some embodiments, the use of extracellular matrix in the culture medium cultures primary state PSCs/ICLCs and 8CLCs. The extracellular matrix is derived from Engelbreth-Holm-Swarm mouse sarcoma (Matrigel ^TM Or Geltrex ^TM Or ECM (electronic program guide) ^TM ) The soluble basement membrane preparation extracted from the plant or the plant comprises human matrix protein collagen IV and at least one component selected from fibronectin, laminin and vitamin C. The extracellular matrix is generally present in the medium of the application in an amount of 0.1% to 0.5% (v/v). If necessary, combinations of different kinds of extracellular matrices may be used, and their total amount in the medium should also be in the range of 0.1% to 0.5% (v/v). Preferably, the extracellular matrix is generally present in the medium of the application in an amount of 0.2% (v/v).

Thus, in one or more preferred embodiments, the culture medium of the application comprises:

(A) DZNep at a final concentration of 5 to 15nM or CPI-1205 at a final concentration of 0.5 to 2mM, and TSA at a final concentration of 3 to 30nM or VPA at a final concentration of 0.25 to 3mM or NaB at a final concentration of 0.25 to 3mM, preferably TSA at a final concentration of 3 to 10nM or VPA at a final concentration of 0.25 to 1mM or NaB at a final concentration of 0.25 to 1 mM; alternatively, a final concentration of 5 to 80nM, preferably 5 to 50nM DZNep or 0.5 to 5mM, preferably 0.5 to 3mM CPI-1205, and a final concentration of 3 to 10nM TSA, or 0.25 to 0.5mM VPA, or 0.25 to 0.5mM NaB;

(B) L-ascorbic acid at a final concentration of 40 to 70 μg/mL;

(C) Human LIF at a final concentration of 10 to 30 ng/mL;

(D) PD0325901 with a final concentration of 0.5 to 1.5 mu M;

(E) IWR1 or XAV939 at a final concentration of 3 to 6. Mu.M; and is further supplemented with:

(1) ACTIVIN a or NODAL at a final concentration of 10 to 25 ng/mL; y27632, thiazovivin or hydroxyfasudil at a final concentration of 0.5 to 2 μm; and 0.1% to 0.5% (v/v) extracellular matrix; or (b)

(2) ACTIVIN a or NODAL at a final concentration of 10 to 25 ng/mL; and a final concentration of 0.5 to 2 μm of Y27632, thiazovivin, or hydroxyfasudil; or (b)

(3) ACTIVIN a or NODAL at a final concentration of 10 to 25 ng/mL; and 0.1% to 0.5% (v/v) extracellular matrix; or (b)

(4) Y27632, thiazovivin or hydroxyfasudil at a final concentration of 0.5 to 2 μm; and 0.1% to 0.5% (v/v) extracellular matrix; or (b)

(5) ACTIVIN a or NODAL at a final concentration of 10 to 25 ng/mL; or Y27632, thiazovivin or hydroxyfasudil at a final concentration of 0.5 to 2. Mu.M; or 0.1% to 0.5% (v/v) extracellular matrix. These media are preferably used to convert primate PSCs to primary PSCs/ICLCs.

More preferably, the medium of the application comprises 10nM DZNep or 1mM CPI-1205;5nM TSA, or 0.5mM VPA, or 0.5mM NaB;50 μg/mL L-ascorbic acid; LIF at 20 ng/mL; 1 μm PD0325901; and 5 μM IWR1 or 5 μM XAV939; and further supplemented with (1) 20ng/mL ACTIVIN A or NODAL,1 μM Y27632, thiazovivin or hydroxyfasudil, and 0.2% (v/v) extracellular matrix; or (2) 20ng/mL ACTIVIN A or NODAL, and 1. Mu.M Y27632, thiazovivin, or hydroxyfasudil; (3) 20ng/mL of ACTIVIN A or NODAL, and 0.2% (v/v) of extracellular matrix; or (4) 1. Mu.M Y27632, thiazovivin or hydroxyfasudil, and 0.2% (v/v) extracellular matrix; or (5) 20ng/mL ACTIVIN A or NODAL, or 1. Mu.M Y27632, thiazovivin or hydroxyfasudil, or 0.2% (v/v) extracellular matrix. These media are preferably used to convert primate PSCs to primary PSCs/ICLCs.

In one or more preferred embodiments, the culture medium of the application comprises DZNep at a final concentration of 40 to 70nM or CPI-1205 at a final concentration of 2 to 4 mM; TSA at a final concentration of 10 to 30nM, or VPA at a final concentration of 0.5 to 1.5mM, or NaB at a final concentration of 0.5 to 1.5 mM; l-ascorbic acid at a final concentration of 40 to 70 μg/mL; LIF at a final concentration of 10 to 30 ng/mL; PD0325901 with a final concentration of 0.5 to 1.5 mu M; and IWR1 or XAV939 at a final concentration of 3 to 6 μm; and is further supplemented with:

(5) ACTIVIN a or NODAL at a final concentration of 10 to 25 ng/mL; or Y27632, thiazovivin or hydroxyfasudil at a final concentration of 0.5 to 2. Mu.M; or 0.1% to 0.5% (v/v) extracellular matrix. These media are preferably used to convert primate PSCs or primary PSCs/ICLCs to 8CLCs.

More preferably, the medium of the application comprises 50nM DZNep or 3mM CPI-1205;20nM TSA, or 1mM VPA, or 1mM NaB;50 μg/mL L-ascorbic acid; LIF at 20 ng/mL; 1 μm PD0325901; and 5 μM IWR1 or 5 μM XAV939; and further supplemented with (1) 20ng/mL ACTIVIN A or NODAL,1 μM Y27632, thiazovivin or hydroxyfasudil, and 0.2% (v/v) extracellular matrix; or (2) 20ng/mL ACTIVIN A or NODAL, and 1. Mu.M Y27632, thiazovivin, or hydroxyfasudil; (3) 20ng/mL of ACTIVIN A or NODAL, and 0.2% (v/v) of extracellular matrix; or (4) 1. Mu.M Y27632, thiazovivin or hydroxyfasudil, and 0.2% (v/v) extracellular matrix; or (5) 20ng/mL ACTIVIN A or NODAL, or 1. Mu.M Y27632, thiazovivin or hydroxyfasudil, or 0.2% (v/v) extracellular matrix. These media are preferably used to convert primate PSCs or primary PSCs/ICLCs to 8CLCs.

In addition to the above components, the culture medium of the application may also comprise other additives commonly used in culture media for stem cells, including but not limited to serum substitutes, such as N2 and/or B27; alternative carbon sources, such as pyruvic acid, e.g. sodium pyruvate; non-essential amino acids; l-glutamine or its substitutes, e.g. Glutamax containing L-alanyl-L-glutamine dipeptide in 0.85% sodium chloride ^TM A supplement; and antibiotics, e.g. panibPenicillin, streptomycin or a mixture of penicillin and streptomycin. The amounts of these additives may be those commonly used for cell culture, in particular stem cell culture.

III method

The media of the invention can be used to reprogram primate somatic cells to primary PSCs/ICLCs, to convert primate PSCs to primary PSCs/ICLCs, and to convert primate PSCs or primary PSCs/ICLCs to 8CLCs.

Accordingly, in one aspect, the invention discloses a method of reprogramming primate somatic cells to primordial state PSCs/ICLCs comprising culturing the somatic cells in a transformation medium comprising a SAH/PRC/EZH2 inhibitor, an HDAC inhibitor, L-ascorbic acid, a JAK/STAT3 signal activator, a MAPK/ERK signal inhibitor and a tankyrase inhibitor, and optionally an ACTIVIN/NODAL signal activator and/or a ROCK inhibitor, with or without extracellular matrix. The thus-produced primary PSCs/ICLCs can be used in a method of converting primary PSCs/ICLCs into 8CLCs. Preferably, the transformation medium is a medium as described in any of the embodiments of the invention.

In another aspect, the invention discloses a method of converting primate PSCs to primary PSC/ICLC, or converting primate PSCs or primary PSCs/ICLCs to 8CLCs, comprising culturing primate PSCs in a conversion medium comprising SAH/PRC/EZH2 inhibitor, HDAC inhibitor, L-ascorbic acid, JAK/STAT3 signal activator, MAPK/ERK signal inhibitor and tankyrase inhibitor, and optionally ACTIVIN/NODAL signal activator and/or ROCK inhibitor, with or without extracellular matrix. In a preferred embodiment, the transformation medium is a medium as described in any of the embodiments of the invention.

In one or more preferred embodiments, the method is a method of converting primate PSCs to pristine PSCs/ICLCs, the conversion medium being a medium having relatively low concentrations of SAH/PRC/EZH2 inhibitor and HDAC inhibitor as defined in any of the embodiments above.

In other preferred embodiments, the method is to convert primate PSCs or naive PSCs/ICLCs to 8CLCs in the medium of any of the above embodiments at relatively high concentrations of the SAH/PRC/EZH2 inhibitor and HDAC inhibitor.

Conventional stem cell culture conditions may be used to convert PSCs into pristine PSCs/ICLCs or 8CLCs. For example, single cell originated PSC can be inoculated into conventional culture medium such as mTESR1 or E8, optionally supplemented with 5-15. Mu.M of ROCK inhibitor such as Y27632. After a period of incubation, such as 24 hours, the medium is changed to the medium of the invention and the cells are continued in the medium until the primary PSCs/ICLCs or 8CLCs are produced. During the cultivation, the fresh medium is preferably changed daily. At passage, the small clones were digested into single cells by conventional methods and, after inoculation, continued to be cultured with the medium of the present invention until either primary PSCs/ICLCs or 8CLCs were formed. Preferably 3-4 days according to 1:4-1:8, and generally, cells can be transformed from primary PSCs/ICLCs for about 2 weeks, cells can be transformed from primary PSCs to 8CLCs for about one week, and cells can be transformed from primary PSCs/ICLCs to 8CLCs in high concentrations of SAH/PRC/EZH2 inhibitor and HDAC inhibitor in 3-5 days. The primary state PSCs/ICLCs for conversion to 8CLC may be primary state PSCs/ICLCs obtained by culturing primate PSCs by any of the methods described herein, or may be known primary state PSCs/ICLCs or primary state PSCs/ICLCs prepared by any of the known methods.

In general, cells can be exposed to normoxic conditions (5% CO at 37℃ ₂ ) Or hypoxia (5% CO) ₂ And 5% O ₂ ) And (5) culturing. The culture time is not particularly limited, and may be easily determined by one skilled in the art based on the present disclosure and conventional techniques in the art. The addition/plating concentration can be determined by one skilled in the art based on common general knowledge in the art and actual production conditions.

In some embodiments of the application, the cells may be cultured under one or more conditions selected from the group consisting of: (i) on feeder cells; (ii) on an extracellular matrix without a feeder layer; (iii) in suspension without feeder cells; (iv) under hypoxic or normoxic conditions at about 37 ℃; (v) Passaging every 3 to 4 days with single cells at a split ratio of 1:4 to 1:8; (vi) daily medium changes.

In some embodiments, to convert primate PSCs to primary PSCs/ICLCs, a single primary primate PSC is added to a feeder layer supplemented with 5 to 15 μm ROCK inhibitor (e.g., Y27632) mTeSR1 or E8 medium for a period of time, e.g., 24 hours, and then the mTeSR1 or E8 medium is replaced with a conversion medium containing relatively low concentrations of SAH/PRC/EZH2 inhibitor and HDAC inhibitor as described herein, and the cells are cultured under hypoxic or normoxic conditions at about 37 ℃ with daily renewal of the medium. During the culture, the cells were passaged every 3 to 4 days at a split rate of 1:4 to 1:8 until the original state PSCs/ICLCs were obtained. In some embodiments, single originating primate PSCs are cultured as described above, but cells are added to the extracellular matrix, e.g., geltrex ^TM On the petri dishes after coating in DMEM-F12, but not on feeder cells.

In some embodiments, to transform primate PSCs to primary PSCs/ICLCs, single, original primate PSCs are added to the plates for a period of time, e.g., 24 hours, using mTeSR1 or E8 medium supplemented with 5 to 15 μm ROCK inhibitor (e.g., Y27632), then the mTeSR1 or E8 medium is replaced with a transformation medium containing relatively low concentrations of SAH/PRC/EZH2 inhibitor and HDAC inhibitor, and the cells are cultured under hypoxic conditions; after forming pellets, transferring the pellets into a flask for suspension culture, and updating the culture medium every day; every 4 to 5 days, single cells were passaged at a split ratio of 1:4 to 1:8 until original PSCs/ICLCs were obtained.

In some embodiments, to convert primate PSCs to 8CLCs, a single original primate PSC is added to a feeder layer of mTeSR1 or E8 medium supplemented with 5 to 15 μm ROCK inhibitor (e.g., Y27632) and incubated for a period of time, e.g., 24 hours, before replacing the medium with a conversion medium containing relatively higher concentrations of SAH/PRC/EZH2 inhibitor and HDAC inhibitor as described herein and culturing the cells under normoxic or hypoxic conditions; every 3 to 4 days, single cells are passaged, and the division ratio is 1:4 to 1:8.

In some embodiments, to convert the primary state PSCs/ICLCs to 8CLCs, single cells are isolated from the primary state PSCs/ICLCs and cultured on a feeder layer with a conversion medium of the application having relatively low concentrations of SAH/PRC/EZH2 inhibitor and HDAC inhibitor for a period of time, e.g., 24 hours, before replacing the medium with a conversion medium of the application having higher concentrations of SAH/PRC/EZH2 inhibitor and HDAC inhibitor as described herein, and culturing for 3 to 5 days without passaging, with daily medium renewal.

In some embodiments, to convert primary PSCs/ICLCs to 8CLCs, single cells are isolated from the primary PSCs/ICLCs and suspended in a conversion medium of the application having relatively low concentrations of SAH/PRC/EZH2 inhibitor and HDAC inhibitor for a period of time that is additionally supplemented with 5 to 15 μm ROCK inhibitor (e.g., Y27632); after formation of small agglomerates, the medium was replaced with the transformation medium of the application with higher concentrations of SAH/PRC/EZH2 inhibitor and HDAC inhibitor without the additional addition of ROCK inhibitor (e.g., Y27632) for several days without passaging, and the medium was refreshed daily.

In some embodiments, teratomas are produced by subcutaneously injecting naive PSCs/ICLCs, 8CLCs, or heavy naive cells as described in any of the embodiments herein into different organs or locations of an immunodeficient mouse or other immunodeficient animal. Organs or locations that may be injected include, but are not limited to, the back, neck, legs, and testes. The immunodeficient mice or other immunodeficient animals include, but are not limited to, immunodeficient mice including, but not limited to, nude mice, SCID mice, NOD-SCID-IL2Rg-/- (NSI) mice, CBA/N mice, beige mice, xid mice, NPG mice, URG mice, NPG-B2M mice, DK-NPg, mice, hIL-3NPG mice, hSCF1 NPG mice, hSCF2 NPG mice, NPG-Fah mice, and F344RG rats. Taking NOD-scid IL2 Rg/immunodeficient mice as an example, one week in advance, mice should be prepared in order to avoid stress. 100 tens of thousands of cells were counted and resuspended in 200ul of a pre-chilled DMEM/F12 and Matrigel1:1 mixture in a 1ml sterile syringe. Mice were fixed from the back and the abdomen was exposed for injection. The needle is inserted obliquely into the skin and transported horizontally to determine the subcutaneous position. The cell suspension is subcutaneously injected into the skin of a male NOD-scid IL2 Rg/mouse with the age of 6-8 weeks, and the injection is slowly withdrawn, so that the cell suspension is prevented from flowing out of the injection site. Teratomas were seen within 3-4 weeks and were collected at 7-8 weeks for subsequent experiments.

In some embodiments, the primary state PSCs/ICLCs, 8CLCs or heavy primary state PSCs described in any of the embodiments herein are allowed to form EB in spheroid form in suspension culture in a self-differentiating medium. In some embodiments, these cells may be seeded onto the plate wall to form spheroids. The plates used to inoculate the cells may be plates well known in the art for EB production including, but not limited to, ultra low adsorption plates (ultralow attachment plate) and AggreWell ^TM A plate. Any known medium that allows self-differentiation can be used in the present application. Culture conditions are well known in the art and are typically at 37℃and 5% CO ₂ Under the condition.

In some embodiments, the original state PSC/ICLC, 8CLC, or heavy original state PSC described in any of the embodiments herein is first cultured to form a uniform EB, and then the EB is cultured in a medium that allows differentiation into the organ of interest, thereby forming the organoid. The EB can be prepared using the methods of preparing EB described in any of the embodiments herein. The organ may be any organ of interest including, but not limited to, brain, liver, kidney, heart, lung, spleen, and intestine. Typically, the original PSC/ICLC, 8CLC or heavy original PSC is cultured in the medium used to form EBs, the medium can be changed daily or every two days, and the resulting EBs transferred to medium allowed to differentiate into organs on about day 6 and cultured for several days, such as for 2-6 days. In some embodiments, cells are seeded into wells of an ultra-low adsorption plate, and suspension cultured in a medium that allows self-differentiation (e.g., mTESR supplemented with Y-27632) to form EBs; after about 3 or 4 days, the medium was replaced with the same medium that was not supplemented with Y-27632; after 1-3 days of culture, EBs are transferred to the ultra low adsorption plates at about day 6 and cultured for several days, such as 2-6 days, in medium that is allowed to differentiate into the organ of interest. The tissue thus formed may be cultured in a differentiation medium suitable for preparing the desired organ, forming aggregates or 3D structures, which culture may be performed on an orbital shaker, e.g. mounted in a culture vessel, which may be rotated at 50-100RPM, to produce the organoids of the organ of interest. As used herein, 3D structures (3D scaffold) include, but are not limited to Matrigel and Geltrex, and the like. The medium that allows differentiation into the organ of interest may be any medium known in the art that differentiates EB into the organ of interest. In some embodiments, for brain organoids, neural induction media and brain differentiation media may be employed. EB medium was changed daily or every two days, and then transferred to neuro-induction medium on day 6 for an additional 4 days of culture. EB will form neuroepithelial tissue. The tissue may be embedded in 3D structures such as Matrigel drops and cultured in brain differentiation medium on an orbital shaker mounted in a culture vessel that may be at a rotational speed of 50-100 RPM. The brain organoids can be collected for later use.

In some embodiments, brain organoids are generated by first seeding 9000 original state PSC, original state PSC/ICLC or 8CLC cells in an ultra-low attached 96-well plate containing EB medium to form uniform EB spheres. EB medium was changed every two days, and the nerve induction culture was renewed on day 6 based on the low adsorption plate and continued for 4 days in the nerve induction medium. EBs will form neuroepithelial tissue, which is then embedded in Matrigel droplets, cultured in brain differentiation medium, and placed on a rail shaker mounted in an incubator at 70 rpm. Brain organoids may be collected on day 30 for further use.

The re-original state PSCs described herein may be obtained by directed differentiation of original state PSCs/ICLCs or 8CLCs using methods commonly used in the art. For example, human ESCs or iPSCs can be cultured in commercially available media such as TeSR or Essenal 8 to obtain heavy, original state PSCs.

The use of any of the transformation media described in any of the embodiments of the present application to reprogram primate somatic cells to primary PSCs/ICLCs, to convert primate PSCs to primary PSCs/ICLCs, or to convert primate PSCs or primary PSCs/ICLCs to 8CLCs, to reprogram primate somatic cells to primary PSCs/ICLCs, or to convert primate PSCs or primary PSCs/ICLCs to 8CLCs, using a medium or a kit, to generate teratomas, organoids or EBs are also included in the present application.

In some embodiments, the application also includes the use of an SAH/PRC/EZH2 inhibitor and an HDAC inhibitor in the preparation of a medium or kit for reprogramming primate somatic cells to iPSCs, or transforming primate PSCs to primary PSCs/ICLCs, or transforming primate PSCs or primary PSCs/ICLCs to 8 CLC. Preferably, the medium or kit may further comprise one or more components selected from the group consisting of L-ascorbic acid, JAK/STAT3 signaling activators, MAPK/ERK signaling inhibitors, and tankyrase inhibitors, and optionally ACTIVIN/NODAL signaling activators and optionally ROCK inhibitors (e.g., Y27632), and optionally extracellular matrix.

In some embodiments, the methods of converting primate PSCs to primary PSCs/ICLCs and vice versa may comprise a genetic engineering step that reduces the SAH, PRC and/or EZH2, and/or HDAC activity of PSCs by knocking down and/or knocking out one or more genes of interest in the cell, and then culturing the engineered PSCs with the media of the application. Preferably, to reduce the activity of SAH, PRC and/or EZH2 of PSCs, the expression of any SAH, PRC and EZH2 modulator can be reduced by knockdown (e.g., siRNA technology), or knockdown (e.g., CRISPR/Cas9 technology). Likewise, expression of HDAC modulators may be reduced in the same manner as described above. After the PSCs are engineered, they can be cultured using the medium of the present application according to the methods described above. In some embodiments, when the SAH, PRC and/or EZH2 activity of PSCs is reduced by engineering, the medium used to culture the engineered PSCs may or may not contain an SAH/PRC/EZH2 inhibitor. Likewise, if the HDAC activity of PSC is reduced by engineering, the culture medium may or may not contain an HDAC inhibitor. In cases where both SAH, PRC and/or EZH2 activity and HDAC activity are reduced as a result of the modification, the medium may contain neither SAH/PRC/EZH2 inhibitor nor HDAC inhibitor, or may contain SAH/PRC/EZH2 inhibitor or HDAC inhibitor.

Thus, in some embodiments, the application further provides a medium comprising neither an SAH/PRC/EZH2 inhibitor nor an SAH/PRC/EZH2 inhibitor or an HDAC inhibitor, or comprising an SAH/PRC/EZH2 inhibitor or an HDAC inhibitor, the remainder of the medium being the same as the composition and amount of the medium described in the second part of the application. In some embodiments, the medium may contain reagents that utilize lipofection. For example, in the above methods, primate PSCs are cultured in a medium comprising liposomes encapsulating a vector of shRNA targeting SAH, PRC and/or EZH2 modulators, which medium comprises, in addition to the vector and the lipid, other components described in part II for the medium of the application, and which medium may or may not comprise an SAH/PRC/EZH2 inhibitor.

IV. Cells

The present application uses to provide human cells/tissues from teratomas, organoids and EBs. Human cells/tissues have transcriptomes similar to human internal cell/tissue types including, but not limited to, airway epithelial cells, epithelial progenitor cells, radial glial cells, cyclic radial glial cells, neurons, melanocytes, mesenchymal stem cells, peri-mesenchymal stem cells, adipogenic mesenchymal stem cells, vascular endothelial cells, smooth muscle cells, peri-cells, fibroblasts, fibroblast progenitor cells, hematopoietic endothelial cells, immune cells, erythrocytes, primitive intestinal endoderm cells, trophoblasts, cytotrophoblasts, villous trophoblasts, placental endothelial cells, granulocyte-macrophage progenitor cells, hematopoietic endothelial cells, mast cells, lymphocytes, neuroblast cells, 5-hydroxytryptamine energy neurons, cyclic neural progenitor cells, granulocyte-progenitor cells, oligodendrocyte precursor cells, schwann precursor cells, endothelial cells, arterial endothelial cells, midgut epithelial cells, postgut epithelial cells, neural stem cells, neuroblast cells, neuroepithelial cells, retinal precursor cells.

The application also provides for the isolation of primate primary PSCs/ICLCs. The primitive PSCs/ICLCs of the application have transcriptomes near the human pre-implantation inner cell mass, transposable element characteristics near the human pre-implantation inner cell mass, DNA methylation groups near the human pre-implantation inner cell mass, chromatin open states near the human pre-implantation inner cell mass, metabolic states near the human pre-implantation inner cell mass.

As used herein, the term "proximal" refers to substantially the same, or not substantially different, and one of ordinary skill in the art will recognize, based on techniques known in the art, that even though some minor differences may exist, the cells of the present application, including cells from the ICLCs and 8 CLCs of the present application, are substantially the same as natural ICM cells or 8 cell embryo cells.

Preferably, pre-implantation ICM marker genes KLF17, DNMT3L, DPPA5, STELLA, TFCP2L1, MAEL and REX1 are significantly induced in the primary PSCs/ICLCs of the present application. More preferably, the expression level of at least one marker gene in the pre-implantation ICM marker genes of the primary PSCs/ICLCs of the application is more than 10 times the expression level of the corresponding pre-implantation ICM marker genes in the primary human PSCs; preferably, the expression level of all of the marker genes is greater than 10 times the expression level of the corresponding pre-implantation ICM marker genes in the original human PSCs.

Preferably, the as-spun PSCs/ICLCs of the present application also have one or more of the following characteristics:

1) Is capable of self-renewal and maintains pluripotency in culture;

2) Maintaining the stability of the genome in culture according to the karyotype;

3) Cells capable of producing 3 germ layers;

4) Capable of producing primordial germ cell-like cells;

5) Can be embedded into mouse embryo and differentiated into embryo and extraembryonic tissue;

6) Can be transformed in vitro to an extraembryonic cell fate; and

7) Can form blastula-like structures in vitro.

Such primary PSCs/ICLCs may be obtained by culturing primate PSCs using any of the methods described in any of the embodiments of the application. Thus, in some embodiments, the application also includes cells obtained by any of the methods described herein, particularly in the pristine state PSCs/ICLCs.

The application also provides isolated 8CLCs that express 8 cell (8C) state-specific marker genes at levels much higher than those of cells in the pre-implantation inner cell mass-like or originating state. In some embodiments, the 8-cell state specific marker genes include ZSCAN4, TPRX1, ZIM3, ZSCAN5B, ZNF280A, and ARGFX. Preferably, the expression level of at least one specific marker gene of the specific marker genes is more than 5 times the expression level of the corresponding 8-cell specific marker gene in the primary PSCs or primary PSCs/ICLCs. Preferably, the expression level of all of the above-mentioned specific marker genes is more than 5 times the expression level of the corresponding 8-cell specific marker genes in the primary PSCs or primary PSCs/ICLCs.

Preferably, the isolated 8CLCs of the application have transcriptomes, transposable element characteristics, and chromatin opening states that approximate human 8-cell embryos. More preferably, the 8CLC of the present application also has one or more of the following features:

1) Maintaining the stability of the genome in culture according to the karyotype;

2) Cells capable of producing 3 germ layers;

3) Capable of producing primordial germ cell-like cells;

4) Can be embedded into mouse embryo and differentiated into embryo and extraembryonic tissue;

5) Can be transformed in vitro to an extraembryonic cell fate; and

6) Can form blastula-like structures in vitro.

The primary state PSCs/ICLCs obtained by somatic reprogramming of somatic cells using the transformation medium of the present application are also within the scope of the present application.

The application also provides cell culture media comprising cells of the application, in particular, the primary PSCs/ICLCs and/or 8CLCs of the application. The cell culture medium may also comprise a culture medium as described in any of the embodiments of the application.

The application is described in the following non-limiting examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present application in any way. Various changes and modifications may be made within the spirit of the application. Unless otherwise indicated, the techniques involved are conventional techniques in various fields of molecular biology, cell biology, biochemistry and the like, which are well known to those skilled in the art.

Example 1: production of primary PSCs/ICLCs and 8CLCs

1. Generation of primary PSCs/ICLCs

Materials and methods

4CL basal medium

1:1 mixture of neural basal medium (Gibco Co.) and higher DMEM/F12 (Gibco Co.), supplemented with N2 supplement (1X, gibco Co.), B27 supplement (1X, gibco Co.), available from N2 and B27, sodium pyruvate (1X, hyclone Co.), non-essential amino acids (NEAA) (Gibco Co.), glutaminase ^TM (1X, gibco Co.) and penicillin-streptomycin (1X, gibco Co.).

4CL supplement

4CL medium 1 was supplemented with 4CL basal medium:

SAH/PRC/EZH2 inhibitor (10 nM DZNep), HDAC inhibitor (5 nM TSA), L-ascorbic acid (50. Mu.g/mL), JAK/STAT3 activator (20 ng/mL human LIF), MAPK/ERK inhibitor (1. Mu.M PD 0325901), tankyrase inhibitor (5. Mu.M IWR 1), ACTIVIN A/NODAL activator (20 ng/mL human ACTIVIN A), extracellular matrix (0.2% (v/v) Geltrex ^TM ) ROCK inhibitor (1 μ M Y27632). Table 1 lists the trademark and catalog numbers of all 4CL supplements.

TABLE 1

Cells

H9 human ESC system

Method

1) Maintenance of originating human PSCs

All ofHuman PSCs provided are routinely stored in Matrigel ^TM Or Geltrex ^TM In mTESR1 or E8 medium on the coated plates. Cells are typically passaged every 4 to 5 days with 0.5mM EDTA. Cells were washed once with PBS at passage and treated with 0.5mM EDTA for 5min. EDTA was then removed and the cells were separated into small pieces using Pasteur pipettes with mTESR1 or E8 medium. Under normoxic conditions (37 ℃, 5% CO) ₂ ) Human PSCs were cultured in an incubator.

2) Conversion to pristine PSCs/ICLCs on feeder layer

The day before the start of transformation, the original human PSCs were washed once with PBS and isolated into single cells at 1000 to 1500 cells/cm ² Is added to the feeder layer supplemented with 10 mu M Y27632 of mTESR1 or E8 medium. After 24 hours, the medium was changed to 4CL medium 1. The same medium was used to renew the medium every 24 hours. Colonies became round and hemispherical in 24 to 48 hours. Cells were passaged every 3 to 4 days. At passage, trypLE was used: 0.5mM EDTA (1:1) separates cells into single cells and at 1000 to 1500 cells/cm ² Is added to the feeder layer (feeder layer is inoculated on Geltrex ^TM /Matrigel ^TM Coated dishes). The original PSCs/ICLC can be induced and maintained at low oxygen (37deg.C, 5% CO) ₂ 、5％O ₂ ) Or normoxic (37 ℃, 5% CO) ₂ 、21％O ₂ ) Under conditions, preferably low oxygen conditions.

2.8CLC Generation

Materials and methods

4CL basal medium

The same as in example 1.

e4CL supplement

e4CL medium was supplemented with 4CL basal medium:

SAH/PRC/EZH2 inhibitor (50 nM DZNep or 3mM CPI-1205), HDAC inhibitor (20 nM TSA or 1mM VPA or 1mM NaB), tankyrase inhibitor (5. Mu.M IWR1 or 5. Mu.M XAV939), L-ascorbic acid (50. Mu.g/mL), JAK/STAT3 activator (20 ng/mL human LIF), MAPK/ERK inhibitor (1. Mu.M PD 0325901), ACTIVIN A/NODAL activator (20 ng/mL human ACTIVIN A or 20ng/mL human NODAL), ROCK inhibitor (1. Mu. M Y2) 7632 or 1. Mu.M Thiazovivin or 1. Mu.M hydroxyfasudil) and extracellular matrix (0.2% (v/v) Geltrex ^TM Or Matrigel ^TM )。

Cells

H9, H1, UH10 human ESC lines.

Method

1) Conversion of originating human PSCs to 8CLCs

The original human PSCs were cultured in the same manner as in example 1. The day before the start of transformation, the cells of the originating human PSCs were isolated as single cells and cultured in mTeSR1 or E8 medium supplemented with 10 μ M Y27632 at 2,000 to 3,000 cells/cm ² Inoculating on the feeding layer. After 24 hours, the medium was changed to e4CL medium at 37℃and 5% CO ₂ Cells are cultured under hypoxic or normoxic conditions. The medium was refreshed daily. The cells were passaged every 3-4 days. At passage, trypLE was used: 0.5mM EDTA (1:1) was dissociated into single cells and at 2000 to 3000 cells/cm ² Is added to the density of the feeder layer coated board. Typically, cells are converted to 8CLCs in about one week.

2) Conversion from original PSCs/ICLCs to 8CLCs

The day before the start of transformation, the primary PSCs/ICLCs were isolated as single cells and cultured in 4CL medium 1 at 2000-3000 cells/cm ² Spread on feeder cells. After 24 hours, the medium was changed to e4CL medium. The medium was refreshed daily. Cells are transformed into 8CLCs in 3-5 days without passaging.

Experimental results

The results are shown in FIGS. 1-6. In FIG. 1, induction of primary PSCs/ICLCs and 8CLCs expressing primary or 8C-specific genes, as evidenced by immunostained images of KLF17 and TPRX1 of either untreated or primary H9 ESCs transformed by 4CL (day 12) or step e4CL (day 5), as shown in FIG. 1 (b); as shown in the heat map of FIG. 1 (c), the heat map is shown in NHSM,Expression of ICM-enriched genes before embryo implantation in human primary PSCs cultured in 5iLAF, 4CL and human ICM cells; the heat map shown in FIG. 1 (d) shows the heat pattern in NHSM, ++>Expression of totipotency genes in 5iLAF or step e4CL (day 5), EPSC and human 8C embryonic cell cultures in the original state ESC; the totipotent gene in the original H9 ESC cultured in direct e4CL (day 7) was verified by RT-qPCR as shown in fig. 1 (e).

The results shown in FIG. 2 demonstrate the matching of the transcriptome of the primary PSCs/ICLCs and 8CLCs with that of the human embryo. Results of return development from human E7 to E3 embryo stages during progressive or direct E4CL induced scRNA-seq time were compared using UMAP, as shown in fig. 2 (a); UMAP visualization of step e4CL-day5 cells as shown in FIG. 2 (b), in which 7 clusters are shown, cluster 5 (8 CLC) accounting for 11.9% of total cell number; by the bubble diagram shown in FIG. 2 (C), the expression frequency and average expression of the representative pluripotency and totipotency genes at the early stage of human embryo, and ESCs in the original state that were not treated or transformed with 4CL (day 8 [ 2 nd generation ] and day 12 [ 3 rd generation ]) and e4CL (day 5C 5[8CLC ] and non-8 CLC [ all other cluster additions ]); by the violin plot shown in fig. 2 (d), there is shown a representative log normalized expression of early human embryo-enriched TE at early stage of human embryo and generation 10 of human ESC compared to the original ESC, 4CL-day 12 original ESC and 8 CLC.

The results shown in FIG. 3 demonstrate that the karyotype of cells induced by primary PSC medium (4 CL) and 8CLC medium (e 4 CL) is normal after long-term culture. Wherein, as shown in FIG. 3 (a), 15 generations of G-banding pattern representative images of the originating H9 and originating iPSC-4 were cultured in 4 CL. Each figure counts 20 cells at metaphase and G-banding pattern representative images of originating H9 and iPSC-4 cultured in step e4CL (day 5) as shown in fig. 3 (b).

The results shown in FIG. 4 demonstrate that cells cultured in the original PSC medium (4 CL) and 8CLC medium (e 4 CL) have lower DNA methylation levels compared to the original PSC. Wherein the violin diagram of FIG. 4 (a) shows the conditions in the original state, 4CL (day 12), 5iLAF,Human PSCs and human 8C embryos and ICMs cultured by NHSM, stepwise e4CL (day 5) and direct e4CL (day 7) pass through global CpG methylation levels measured by RRBS; in FIG. 4 (b), it is shown that in the original state, 4CL (day 12), 5iLAF,/is in the original state>CpG methylation levels of human PSCs, ICMs and post-implantation embryo imprinting control areas cultured under NHSM and stepwise e4CL (day 5) conditions; genome-map trace shows that in the condition of origin, 4CL (day 12), 5iLAF,/I >CpG methylation levels of PSCs cultured under NHSM, stepwise e4CL (day 5) and direct e4CL (day 7), human 8C embryos and ICM indicated the original state multipotent (blue) and totipotent (red) sites as shown in fig. 4 (C).

The results shown in FIGS. 5 (a) - (d) demonstrate chromatin accessibility of primary PSC/ICLC and 8CLC to human embryo matches. Wherein UMAP gene scores of all genes in scaTAC-seq are visualized, originating ESCs untreated (red), 4CL (day 12; blue) or stepwise e4CL (day 5; green), as shown in FIG. 5 (a); UMAP visualization based on FIG. a, highlighting the gene scores for the originating state (ZIC 2), shared primitive state pluripotency/8 CLC (DPPA 3) and the totipotency genes expressed in each cell in the originating state ESCs untreated or transformed by 4CL (day 12) and step e4CL (day 5), as shown in FIG. 5 (b); genome reader tracking showed chromatin accessibility, H3K27ac levels and transcription factor DNA binding motif positions at the original state multipotent KLF17 and totipotent ZSCAN4 sites.

The results shown in FIG. 6 demonstrate that 8CLC was elevated by sorting TPRX1-GFP reporter cells. As shown in the upper panel of FIG. 6 (a), EGFP was inserted into the TPRX1 site of H9 ESCs or HN10-DsRed ESCs (for chimeric experiments), and donor constructs for generating TPRX1-EGFP reporter cell lines. The lower panel shows the results of knocking TPRX1-EGFP into cells and verifying them by e4CL step culture (day 5) and anti-TPRX1 immunostaining with GFP ⁺ Signal agreement (left panel). TPRX1-EGFP cells in step e4CL (day 5)FACS analysis showed GFP ⁺ Percentage of cells (right panel). As shown in the bubble diagram of fig. 6 (b), the expression frequency and average expression of genes representing pluripotency and totipotency at the early embryo stage of human and the 10 th generation of human ESC are represented compared to the original ESC, 4CL original ESC and sorted 8CLC at day 12.

Example 2: production of pristine PSCs/ICLCs by ESCs or iPSCs

Materials and methods

4CL basal medium

The same as in example 1.

4CL supplement

The same as in example 1.

Cells

Human ESC lines: h1 (male), HN10 (female), HUES1 (male), and WIBR3 (female); human iPSC line: CBC14 (inventor made, female), C11 (inventor made, female), phoenix (Ulrich Martin laboratory gift, female), diPS 1016SevA (purchased from harvard stem cell institute, male), STiPS O-XX1 (purchased from harvard stem cell institute, female), UH10 (male).

Method

The same method as in example 1 was used.

Experimental results

The RT-qPCR data in fig. 7 shows that the pre-implantation ICM marker genes KLF17, DNMT3L, DPPA5, STELLA, TFCP2L1, KLF4, MAEL and REX1 were significantly induced from the original state PSC/ICLC transformed from various original state human PSC lines. The 4CL Medium 1 proved to have broad applicability for induction of human PSC.

Example 3: production of pristine PSCs/ICLCs in extracellular matrix

Materials and methods

4CL basal medium

The same as in example 1.

4CL supplement

The same as in example 1.

Cells

H9 human ESC lines.

Method

Use and implementationExample 1 the same procedure was followed except that the cells were added to a cell containing 1% (v/v) Geltrex ^TM DMEM-F12 (cat#) coated on dishes instead of feeder cells.

Experimental results

FIG. 8 shows RT-qPCR data in medium 1 containing 4CL, geltrex ^TM Of the primary PSCs/ICLCs transformed on the coated dishes, pre-implantation ICM marker genes KLF17, DNMT3L, DPPA, STELLA, TFCP2L1, KLF4, MAEL and REX1 were significantly induced, similar to the primary PSCs/ICLCs on the feeder layer. Demonstrating that 4CL medium 1 was also effective without feeder cells.

Example 4: production of as-spun PSCs/ICLCs in suspension

Materials and methods

4CL basal medium

The same as in example 1.

4CL supplement

The same as in example 1.

Cells

H9 human ESC lines.

Method

The original human PSCs were cultured in the same manner as in example 1. The day before the start of transformation, the originating human PSCs cells were isolated as single cells and added to aggresell at 60,000 cells/well with mTeSR1 or E8 medium supplemented with 10 μ M Y27632 ^TM 800 plates. After 24 hours, the medium was changed to 4CL medium 1, and then to hypoxia culture. Cells formed pellets within 3 days. These pellets were then resuspended and transferred to a low adsorption flask (Greiner Bio One, 658190) for suspension culture. The medium was refreshed daily. The cells were passaged every 4-5 days. At passage, cells were isolated as single cells using TrypLE:0.5mM EDTA (1:1) and resuspended in 4CL Medium 1 at a density of 150,000 cells/mL. The resuspended cells were then added to a low adsorption flask (Greiner Bio One, 658190) for suspension culture. Cells formed small aggregates within 24 hours. Typically, cells are transformed to pristine PSCs/ICLCs approximately 3 weeks after initiation.

Experimental results

FIG. 9 is RT-qPCR data showing that pre-implantation ICM marker genes KLF17, DNMT3L, DPPA5, STELLA, TFCP2L1, KLF4, MAEL and REX1 were significantly induced in primary PSCs/ICLCs suspension transformed with 4CL Medium 1. The 4CL Medium 1 was shown to be as effective for suspension culture.

Example 5: generation of as-spun PSCs/ICLCs in 4CL without ECM/ROCK inhibitor/ACTIVIN/NODAL activator

Materials and methods

4CL basal medium

The same as in example 1.

4CL supplement

4CL medium 2 (minus extracellular matrix) was supplemented in 4CL basal medium:

SAH/PRC/EZH2 inhibitor (10 nM DZNep), HDAC inhibitor (5 nM TSA), L-ascorbic acid (50 μg/mL), JAK/STAT3 activator (20 ng/mL human LIF), MAPK/ERK inhibitor (1 μM PD 0325901), tankyrase inhibitor (5 μM IWR 1), ACTIVIN A/NODAL activator (20 ng/mL human ACTIVIN A), and ROCK inhibitor (1 μ M Y27632).

4CL medium 3 (minus ROCK inhibitor) was supplemented in 4CL basal medium:

SAH/PRC/EZH2 inhibitor (10 nM DZNep), HDAC inhibitor (5 nM TSA), L-ascorbic acid (50. Mu.g/mL), JAK/STAT3 activator (20 ng/mL human LIF), MAPK/ERK inhibitor (1. Mu.M PD 0325901), tankyrase inhibitor (5. Mu.M IWR 1), ACTIVIN A/NODAL activator (20 ng/mL human ACTIVIN A), extracellular matrix (0.2% (v/v) Geltrex ^TM )。

4CL medium 4 (minus ACTIVIN/NODAL activator) was supplemented in 4CL basal medium:

SAH/PRC/EZH2 inhibitor (10 nM DZNep), HDAC inhibitor (5 nM TSA), L-ascorbic acid (50 μg/mL), JAK/STAT3 activator (20 ng/mL human LIF), MAPK/ERK inhibitor (1 μM PD 0325901), tankyrase inhibitor (5 μM IWR 1), extracellular matrix (0.2% (v/v) Geltrex ^TM ) And ROCK inhibitors (1 μ M Y27632).

Cells

H9 human ESC lines.

Method

The same method as in example 1 was used.

Experimental results

FIG. 10 is RT-qPCR data showing that pre-implantation ICM marker genes KLF17, DNMT3L, DPPA5, STELLA, TFCP2L1, KLF4, MAEL and REX1 were significantly induced in the original state PSCs/ICLCs transformed with 4CL Medium 2 (A panel), 4CL Medium 3 (B panel), 4CL Medium 4 (C panel), respectively. These results indicate that it does not contain Geltrex ^TM 4CL Medium, ROCK inhibitor or ACTIVIN/NODAL activator is also effective.

Example 6: generation of 8CLC using male or female cell lines

Materials and methods

4CL basal medium

The same as in example 1.

e4CL supplement

e4CL medium was supplemented in 4CL basal medium:

SAH/PRC/EZH2 inhibitor (50 nM DZNep or 3mM CPI-1205), HDAC inhibitor (20 nM TSA or 1mM VPA or 1mM NaB), L-ascorbic acid (50 μg/mL), JAK/STAT3 activator (20 ng/mL human LIF), MAPK/ERK inhibitor (1 μM PD 0325901), tankyrase inhibitor (5 μM IWR1 or 5 μM XAV939), ACTIVIN A/NODAL activator (20 ng/mL human ACTIVIN A or 20ng/mL human NODAL), ROCK inhibitor (1 μ M Y27632 or 1 μM Thiazovivin or 1 μM hydroxyfasudil) and extracellular matrix (0.2% (v/v) Geltrex ^TM Or Matrigel ^TM )。

Cells

H9, H1, UH10 human ESC lines.

Method

1) Conversion from originating human PSCs to 8CLCs

The original human PSCs were cultured in the same manner as in example 1. The day before the start of transformation, the cells of the originating human PSCs were isolated as single cells and cultured in mTeSR1 or E8 medium supplemented with 10 μ M Y27632 at 2,000 to 3,000 cells/cm ² Adding the mixture onto the feeder layer. After 24 hours, the medium was changed to e4CL medium at 37℃with 5% CO ₂ Cells are cultured under hypoxic or normoxic conditions. The medium was refreshed daily. The cells were passaged every 3-4 days.At passage, cells were isolated into single cells using TrypLE:0.5mM EDTA (1:1) and at 2000 to 3000 cells/cm ² Is added to the density of the feeder layer coated board. Typically, cells are converted to 8CLCs in about one week.

2) Conversion from original PSCs/ICLCs to 8CLCs

The day before the start of transformation, ICLC was isolated as single cells and cultured in 4CL Medium 1 at 2000-3000 cells/cm ² Loaded onto the feeder layer. After 24 hours, the medium was changed to e4CL medium. The medium was refreshed daily. Cells are transformed into 8CLC in 3-5 days without passage.

Experimental results

FIG. 11RT-qPCR data shows that human 8C-specific marker genes ZCAN 4, TPRX1, ZIM3, ZCAN 5B, ZNF280A and ARGFX were significantly induced in 8CLCs transformed from either human-originated PSCs or primary PSCs/ICLCs.

Example 7: production of 8CLC in suspension

Materials and methods

4CL basal medium

The same as in example 1.

e4CL supplement

The same as in example 6.

Cells

H9 human ESC lines.

Method

Conversion from suspension cultured primary PSCs/ICLCs to suspension cultured 8CLCs

The original PSCs/ICLCs were cultured in the same manner as in example 1. The day before the start of transformation, the original state PSCs/ICLCs were isolated as single cells and resuspended in 4CL Medium 1 at a density of 300000 cells/ml. The cell suspension was then added to a low adsorption flask (Greiner Bio One, 658190) for suspension culture. After 24 hours, the cells formed small aggregates and the medium was changed to e4CL medium. The medium was refreshed daily and cells transformed to 8CLC in 3 to 5 days without passaging.

Experimental results

The RT-qPCR data in fig. 12 shows that in 8CLC suspension transformed with e4CL medium, the 8C marker genes ZSCAN4, ARGFX, TPRX1, ZNF280A and ZSCAN5B were significantly induced. The e4CL medium was shown to be as effective for suspension culture.

Example 8: production of 8CLC by ESC and iPSC

Materials and methods

4CL basal medium

The same as in example 1.

e4CL supplement

The same as in example 6.

Cells

Human ESC lines: HN10 and UH10

Method

The same as in example 6.

Experimental results

The RT-qPCR data of fig. 13 shows that 8C marker genes ZSCAN4, ARGFX, TPRX1, ZNF280A, ZSCAN5B, DUXA, DUXB, MBD L2, STELLA, KLF17 and KHDC1L were significantly induced in 8CLCs transformed from various human PSCs lines. The e4CL medium was shown to have wide applicability to human PSC.

Example 9: teratoma production

Materials and methods

DMEM/F12，

And (3) cells:

human 8CLC, original PSC/ICLC from examples 1-8 or reinitiated PSC obtained from human ESCs cultured in mTESR 1.

Method

1) Animal preparation

Male NOD-scid-IL2 Rg-/-mice were maintained in the SPF facility environment for 6-8 weeks of age.

2) Preparation of original PSC, original PSC/ICLC and 8CLC suspension

100 ten thousand original PSCs, original PSC/ICLC or 8CLC were isolated from the medium, collected with 200. Mu.L of 1:1DMEM/F12 andand re-suspending the pre-cooling liquid. The heavy suspension is placedOn ice until implantation. The transplantation should be performed immediately.

3) Transplantation

The original PSC/ICLC or 8CLC suspensions were collected in 1ml syringes and subcutaneously injected into 6-8 week old male NOD-scid-IL2 Rg-/-mice.

To avoid stress, the mice need to be primed at least one week in advance. At teratoma formation, 100 ten thousand cells were counted in a 1mL sterile syringe with 200. Mu.l of a pre-chilled 1:1DMEM/F12 and Matrigel mix. Mice were fixed from the back and the abdomen was exposed for injection. The needle is inserted obliquely into the skin and operated horizontally to determine the subcutaneous position. The cell suspension is injected into the subcutaneous of a male NOD-scid IL2 Rg-/-mouse with the age of 6 to 8 weeks, and the needle is slowly pushed out, so that the cell suspension is prevented from flowing out of an injection position.

4) Animal monitoring during teratoma formation

Mice were maintained on SPF-grade facilities for 8 weeks after injection. The size of teratomas grown at the injection site was visually observed. Typically, teratomas are isolated 6-8 weeks after injection.

5) Collecting human cells from teratomas

Mice were sacrificed 8 weeks after injection and teratomas were isolated. The collected teratomas are used for frozen sections, cell isolation, single cell transcriptome analysis, or other uses.

Experimental results

The results shown in FIG. 14 indicate that cells in the original state, PSCs/ICLCs (4 CL) and 8CLC, form teratomas. As shown in fig. 14 (a), representative images of teratomas were derived from starting ESCs after sorting transformation with 4CL (15 th generation) and 8 CLC; hematoxylin and eosin staining patterns as shown in fig. 14 (b) are originating ESC teratomas transformed with 4CL (15 th generation) and 8CLC after sorting. A tissue representative image corresponding to three germ layers is shown; as shown in fig. 14 (c), UMAP visualization showed cell types identified in 8clc, e4CL-day5 cells, 4CL naive ESC and originating ESC teratoma scRNA-seq from sorting. H9ESC was used to generate these teratoma cell types.

The results shown in figure 15 are teratoma cell annotations for different starting cell types. UMAP visualization based on FIG. 14c, cell types identified in the scRNA-seq dataset generated from the original ESCs, 4CL original PSCs, step e4CL-day5 cells and sorted 8 CLCs are shown.

The results shown in fig. 16 represent teratoma cell type quantification. The naive PSC/ICLC teratomas or 8CLC teratomas can be derived from embryos (3-germ layer cell types) and extraembryonic trophoblasts, with the bar graph shown in FIG. 16 (a) showing the distribution of different teratomas to identified cell types and the bar graph shown in FIG. 16 (b) showing the relative distribution of different teratomas to each embryo (ectodermal, mesodermal, and endodermal) and extraembryonic (trophoblast) lineages.

The results shown in fig. 17 (a) to 17 (d) indicate that teratoma trophoblast subtype annotation and quantification, teratoma trophoblast can further produce cytotrophoblast, villous trophoblast and placental endothelial cells; FIG. 17 shows UMAP visualization of identification of cell types from scRNA-seq of teratomas generated from sorted 8CLC, e4CL-day 5 cells, 4 CL-naive ESCs and naive ESCs, respectively, and results of frequency and average expression levels of marker genes in the extra-embryonic trophoblast lineage cell subtypes, with ordered 8CLC, e4CL-day 5 cells, pristine ESCs and naive ESCs producing a histogram of the relative distribution of teratomas to the extra-embryonic trophoblast lineage cell subtypes and UMAP visualization showing the distribution and expression of relevant markers in the indicated teratoma trophoblast cells according to FIG. 14 c.

Fig. 18 shows annotation and quantification of teratoma immune cell subtypes, including granulocyte-macrophage progenitor cells, hematopoietic endothelial cells, mast cells, lymphocytes, and erythrocytes. UMAP visualization shows labeled immune cell subsets (left panel) and primary PSCs, 4CL naive ESCs, step e4CL-day-5 cells and sorted 8 CLC-derived teratoma cells (right panel).

b. Bubble figures represent the frequency and average expression level of marker genes in different immune cell subtypes.

c. Bar graphs show the distribution of sorted 8CLC, stepwise e4CL-day-5, 4 CL-primary and primary PSC-derived teratomas for different immune cell subtypes.

Fig. 19 is an illustration and quantification of teratoma neuronal cell subtypes, including circulating eomes+ intermediate progenitor cells, dopaminergic neuron progenitor cells, dopaminergic neurons, gama-aminobutyric acid energy neurons, glutamatergic neurons, immature neurons, neuroblast cells, radioglia, 5-hydroxytryptamine energy neurons.

UMAP visualization shows distribution of teratoma cells obtained from primary PSC,4CL primary PSC, e4CL cells and sorted 8 CLC;

UMAP visualization shows annotated sub-clusters of neuronal cells from primary PSC,4CL primary PSC, e4CL cells and sorted 8 CLC-derived malformations;

c. Bar graphs show the relative contributions of different neuronal cell types to the indicated teratomas;

d. bar graphs show the contributions of different teratomas to the identification of cell types.

Example 10: generation of brain organoids

Materials and methods

DMEM-F12 (Invitrogen company cat.no.11330-032or 31330-038,depending on location)

Nerve basic culture medium (Gibco company)

Glutaminase TM (Invitrogen cat.no.35050-038)

Non-essential amino acids (Sigma cat.no.M7145)

Penicillin-streptomycin (Sigma Co)

N2 supplement (Invitrogen corporation)

B27 supplement contains no vitamin a (Invitrogen cat.no.12587010)

B27 supplement contains vitamin a (Invitrogen cat.no.17504044)

Y27632 (Axon Medchem 1681)

Mercaptoethanol (Merck company cat.no.8057400005)

Heparin (Sigma cat.no.H3149)

Insulin (Sigma cat.no. I9278-5 ML)

Brain-derived neurotrophic factor (R & D company 248-BDB-250)

Matrigel minus growth factor (BD Biosciences company 356230)

U type bottom low adsorption 96 well plate (Corning company cat.no. CLS 7007)

Low adsorption 24 orifice plate (Corning company cat.no.CLS3473)

60X 15mm Low adsorption tissue culture dish (Corning company cat.No.3261)

Culture medium

Cells

Human 8CLC in example 1, either original PSC/ICLC or re-original PSC was obtained from human ESCs cultured in mTESR 1.

Method

Day 0

At a cell growth ratio of 70-80% of the plate area, clones were digested to single cells with accutase and resuspended in 150 μl EBM for 9000 cells/well to be inoculated into 96-well low-adsorption plates.

Day 1

After 24h, small embryoid bodies with clear boundaries can be observed under a tissue culture microscope at 37℃and 5% CO ₂ Continuously culturing embryoid bodies in a cell culture incubator.

Day 2

Old medium was removed to avoid touching embryoid bodies at the bottom of the plate, and 150 μl of fresh EBM was added to the plate.

Day 4

Fresh EBM without Y was added to the plates.

Day 6

The embryoid body was transferred to a 24-well low suction plate with a 200. Mu.L flaring gun head and 500ul of NIM was added thereto.

Day 8

500ul of fresh NIM was added to the plates.

Day 10: transferring neuroepithelial-like tissue mass in Matrigel drops

Matrigel was dissolved at 4 ℃ for 30 minutes.

2. The sealing film was placed on a 200. Mu.L-sized gun-loading head with an area of (4 x 4) 16 holes and pressed with a finger to form a small depression. Ultraviolet sterilization is carried out for 30 minutes.

3. The neuroepithelial-like tissue mass was transferred to each small well prepared in advance with a 200 μl flaring gun head.

4. Excess medium in each well was carefully removed with a 200. Mu.L tip.

5. Immediately, a volume of about 30. Mu.L of growth factor-reduced Matrigel was added dropwise to the tissue mass in each well to fill the well.

6. The neuroepithelial-like tissue mass was dialed to the center of the droplet with a 10 μl gun head.

7. Place the groove set at 60mm ² In a culture dish, incubated at 37℃for 20-30 minutes in order to allow the Matrigel droplets to pack the tissue mass.

8. To 60mm ² 5mL of CDM containing no vitamin A was added to the culture dish.

9. The sealing membrane was turned using sterile forceps and Matrigel droplets were detached from the sealing membrane by shaking the petri dish. If the liquid drops still remain on the sealing film, one end of the sealing film can be fixed by forceps and the sealing film is rocked in the culture medium, so that the residual liquid drops fall off. The detached Matrigel droplets were placed in a cell incubator for continuous culture.

Day 12

To the culture dish was added 5mL of CDM without vitamin a.

Day 14

To the culture dish, 5mL of CDM containing vitamin A was added, and the dish was transferred to an incubator equipped with a shaking table at 70rpm, and the medium was changed every 3 to 4 days.

Day 30

To the culture dish, 5mL CDM containing 14ng/mL BDNF was added, and the medium was changed every 3-4 days.

Experimental results

FIG. 20 shows annotation and quantification of brain organoid cell types, including cyclic neural progenitor cells, granulocyte-macrophage progenitor cells, oligodendrocyte precursor cells, radial glial cells, and schwann cell precursor cells.

UMAP visualization shows the proportion of brain organoids derived from PSCs in the as-initiated and 4CL as-initiated states;

umap visualization shows annotated subpopulations of nerve cells in brain organoids derived from PSCs in the as-initiated and 4CL as-initiated states;

c. bar graphs show the ratio of each cell type in the two brain organoids;

d. bar graphs show the proportion of the two brain organoids in each cell type.

Example 11: generation of embryoid bodies

Materials and methods

Accutase

AggreWell ^TM plate

10cm of low adsorption ² Culture dish

EB differentiation medium

Cells

Human 8CLC and original PSC/ICLC in example 1 or re-original PSC was obtained by incubating human ESCs in mTESR 1.

Method

Clones were digested with accutase to single cells when the growth ratio of primary PSCs/ICLCs and 8CLCs cells was 70-80% of the area of the plates. 6X 10 ⁵ Individual cells were resuspended in 2ml of EB differentiation medium containing Y-27632 and inoculated into 24-well AggreWell ^TM In the plate, aggreWell was immediately started ^TM Plates were centrifuged at 100g for 3 min to trap cells in the microwells and observed under a microscope to ensure uniform cell distribution in the microwells. At 37 ℃,5% CO ₂ And culturing for 24 hours under the condition of 95% humidity, and observing under a mirror. 1-1.5mL of medium was slowly aspirated and 1.5mL of fresh EB differentiation medium was added along the wall to avoid EB pellet loss. After 2-4 days of continued culture, the floating EB pellet was either suspended for 20 days (method 1) or after 20 days of suspension culture, gelatin coated, before transferring the floating EB pellet to a low adsorption plate for sample harvesting Samples were harvested after additional 15 days of adherent differentiation on the plates. The sample is used for subsequent cell sorting sequencing or other uses.

EB acquisition

The EB pellet was transferred to a 15mL or 50mL centrifuge tube using a papanicolaou dropper. EB beads were obtained by pipetting the plate bottom with 1mL of warm medium. The EB pellet was transferred 3 times repeatedly into the same centrifuge tube. The centrifuge tube remained stable to allow the EB spheres to settle sufficiently. The upper medium was gently removed. Add 2ml DPBS to the centrifuge tube, carefully mix the EB pellet, hold the centrifuge tube steady for 5 minutes to bottom the EB pellet and gently remove the upper DPBS. The wash was repeated once and EBs digested for subsequent study.

Experimental results

FIG. 21 shows annotation and quantification of EB cell types, including endodermal epithelial cells, endothelial cells, arterial endothelial cells, midgut epithelial cells, hindgut epithelial cells, neural stem cells, neuroblast cells, neuroepithelial cells, retinal precursor cells, schwann cell precursor cells, smooth muscle cells, and trophoblast cells. Wherein the UMAP visualization shown in FIG. 21 (a) shows the distribution of EB different embryo layer cells resulting from PSCs in the original state and the 4CL original state; FIG. 21 (b) shows UMAP visualization showing annotated cell types in EBs derived from PSCs in the as-initiated and 4CL as-initiated states; FIG. 21 (c) is a bar graph showing the duty cycle of different cell types in a given EB; the bar graph shown in fig. 21 (d) shows the duty cycle of different EBs in a given cell type.

Claims

1. A method of producing teratomas, said method comprising the steps of transplanting primary, originating, 8clc or heavy primary PSCs into different organs or locations of an immunodeficient animal of interest and feeding said animal;

wherein the method of making the primary PSCs/ICLCs comprises the step of culturing primate PSCs in a medium comprising an SAH/PRC/EZH2 inhibitor, an HDAC inhibitor and a WNT/beta-catenin signaling inhibitor; the preparation method of the 8CLCs comprises the steps of culturing primate PSCs or the original PSC/ICLC in a culture medium containing optimized doses of SAH/PRC/EZH2 inhibitor, HDAC inhibitor and WNT/beta-catenin signal inhibitor; the heavy original state PSCs are obtained through induced differentiation of original state PSCs/ICLCs or 8 CLCs.

2. A method of producing organoids, said method comprising the step of suspension culturing primary, 8clc or heavy primary PSCs, or culturing said primary, 8clc or heavy primary PSCs on a 3D scaffold in a medium that allows differentiation into a target organ; wherein the method of preparing the primary PSCs/ICLCs comprises the step of culturing primate PSCs in a medium comprising an SAH/PRC/EZH2 inhibitor, an HDAC inhibitor and a WNT/beta-catenin signal inhibitor; the preparation method of the 8CLCs comprises the steps of culturing primate PSCs or original PSCs/ICLCs in a culture medium containing optimized doses of SAH/PRC/EZH2 inhibitor, HDAC inhibitor and WNT/beta-catenin signal inhibitor; the heavy original state PSCs are obtained by inducing differentiation of original state PSCs/ICLCs or 8 CLCs.

3. A method of producing embryoid bodies, comprising the step of suspension culturing said primary, 8clc or heavy primary PSCs in a medium that allows differentiation into a target organ; wherein the method of preparing the primary PSCs/ICLCs comprises the step of culturing primate PSCs in a medium comprising an SAH/PRC/EZH2 inhibitor, an HDAC inhibitor and a WNT/beta-catenin signal inhibitor; the preparation method of the 8CLCs comprises the steps of culturing primate PSC or primary PSC/ICLC in a culture medium containing optimized doses of SAH/PRC/EZH2 inhibitor, HDAC inhibitor and WNT/beta-catenin signal inhibitor; the heavy original PSCs are obtained by inducing differentiation of original PSCs/ICLCs or 8 CLCs.

4. The method of any one of claims 1-3, wherein the medium is further supplemented with one or more of L-ascorbic acid or a derivative thereof, a JAK/STAT3 signaling activator, and a MAPK/ERK signaling inhibitor;

optionally, the medium is further supplemented with one or more of an ACTIVIN/NODAL signal activator, a ROCK inhibitor, and an extracellular matrix.

5. The method according to any one of claims 1 to 4, wherein,

The PRC/EZH2 inhibitor or SAH inhibitor is selected from DZNep and CPI-1205; the final concentration of DZNep in the medium is preferably 5-80nM, more preferably 5-50nM; the final concentration of CPI-1205 in the medium is preferably 0.5-5mM, more preferably 1-3mM; and/or

The HDAC inhibitor is selected from TSA, VPA and NaB; preferably, the final concentration of TSA in the medium is 3-30nM, more preferably 3-25nM; preferably, the final concentration of VPA in the medium is 0.25-2mM, more preferably 0.5-1.5mM; preferably, the NaB is present in the medium at a medium concentration of 0.25-2mM, more preferably 0.5-1.5mM; and/or, preferably, the final concentration of the WNT/beta-catenin signal inhibitor in the medium is 2 to 8. Mu.M; preferably, the WNT/beta-catenin signal inhibitor is selected from IWR1 and XAV939.

6. The method according to claim 4, wherein:

the final concentration of L-ascorbic acid in the culture medium is 40-70 μg/mL, and/or

The final concentration of the JAK/STAT3 signal activator in the culture medium is 10-50ng/mL; preferably, the JAK/STAT3 signaling activator is LIF; and/or

The final concentration of the MAPK/ERK signal inhibitor in the culture medium is 0.5-3 mu M; preferably, the MAPK/ERK signaling inhibitor is PD0325901; and/or

The final concentration of the ACTIVIN/NODAL signal activator is 10-25ng/mL; preferably, the activator of the ACTIVIN/NODAL signal is selected from ACTIVIN a and NODAL; and/or

The final concentration of the ROCK inhibitor is 0.5-2 mu M; preferably, the ROCK inhibitor is selected from Y27632, thiazovivin, and hydroxyfasudil; and/or

The extracellular matrix is contained in the culture medium in an amount of 0.1-05% (v/v); preferably, the extracellular matrix is selected from Matrigel ^TM 、Geltrex ^TM And ECM ^TM 。

7. A method according to any one of claims 1-3, wherein the medium used to prepare the pristine PSCs/ICLCs comprises:

(A) DZNep at a final concentration of 5-15nM or CPI-1205 at a final concentration of 0.5-2mM, TSA at a final concentration of 3-30nM or VPA at a final concentration of 0.25-2mM or NaB at a final concentration of 0.25-2mM, preferably TSA at a final concentration of 3-10nM or VPA at a final concentration of 0.25-1mM or NaB at a final concentration of 0.25-1 mM; or DZNep at a final concentration of 5-80nM, preferably 5-50nM or CPI-1205 at a final concentration of 0.5-5mM, preferably 0.5-3mM, and TSA at a final concentration of 3-10nM or VPA at a final concentration of 0.25-0.5mM or NaB at a final concentration of 0.25-0.5 mM;

(B) L-ascorbic acid with a final concentration of 40-70 mug/mL;

(C) LIF with a final concentration of 10-30 ng/mL;

(D) PD0325901 with a final concentration of 0.5-1.5 mu M;

(E) IWR1 or XAV939 at a final concentration of 3-6. Mu.M;

the medium further comprises:

8. The method of claim 7, wherein the step of determining the position of the probe is performed,

the medium included 10nM DZNep or 1mM CPI-1205;5nM TSA, or 0.5mM VPA, or 0.5mM NaB;50 μg/mL L-ascorbic acid; 20ng/mL LIF;1 μM PD0325901; and 5 μM IWR1 or 5 μM XAV939;

And further supplement:

(1) 20ng/mL ACTIVIN A or NODAL, 1. Mu.M Y27632, thiazovivin or hydroxyfasudil, and 0.2% (v/v) extracellular matrix; or (b)

(2) 20ng/mL ACTIVIN A or NODAL, and 1. Mu.M Y27632, thiazovivin or hydroxyfasudil;

(3) 20ng/mL ACTIVIN A or NODAL, and 0.2% (v/v) extracellular matrix; or (b)

(4) 1. Mu.M of Y27632, thiazovivin or hydroxyfasudil, and 0.2% (v/v) of extracellular matrix; or (b)

(5) 20ng/mL ACTIVIN A or NODAL, or 1. Mu.M Y27632, thiazovivin or hydroxyfasudil, or 0.2% (v/v) extracellular matrix.

9. A method according to any one of claims 1-3, wherein the medium used to prepare 8CLCs contains a final concentration of 40-70nM of DZNep or a final concentration of 2-4mM of CPI-1205; TSA at a final concentration of 10-30nM, or VPA at a final concentration of 0.5-1.5mM or NaB at a final concentration of 0.5-1.5 mM; l-ascorbic acid with a final concentration of 40-70 mug/mL; LIF with a final concentration of 10-30 ng/mL; PD0325901 with a final concentration of 0.5-1.5 mu M; and IWR1 or XAV939 at a final concentration of 3-6. Mu.M, respectively; and further supplement:

10. The method of claim 9, wherein the medium comprises 50nM DZNep or 3mM CPI-1205;20nM TSA, or 1mM VPA, or 1mM NaB;50g/mL L-ascorbic acid; LIF at 20 ng/mL; 1 μM PD0325901; and 5 μM IWR1 or 5 μM XAV939;

and further supplement:

(1) 20ng/mL ACTIVIN A or NODAL,1 μ M Y27632, thiazovivin or hydroxyfasudil, and 0.2% (v/v) extracellular matrix; or (b)

(2) 20ng/mL ACTIVIN A or NODAL, and 1 μ M Y27632, thiazovivin or hydroxyfasudil;

(3) 20ng/mL ACTIVIN A or NODAL, and 0.2% (v/v) extracellular matrix; or (b)

(4) 1 μ M Y27632, thiazovivin or hydroxyfasudil, and 0.2% (v/v) extracellular matrix; or (b)

(5) 20ng/mL ACTIVIN A or NODAL, or 1 μ M Y27632, thiazovivin or hydroxyfasudil, or 0.2% (v/v) extracellular matrix.

11. The method according to any one of claims 1-10, wherein the basal medium of the medium used to prepare the original PSCs/ICLCs and 8CLCs is selected from Dulbecco's Modified Eagle Medium (DMEM), minimal Essential Medium (MEM), basal Medium Eagle (BME), RPMI1640, F10, F12, alpha minimal essential medium (alpha MEM), glass Minimal Essential Medium (GMEM), iscove's modified Dulbecco's medium, neural basal medium, DMEM/F12 and advanced DMEM/F12 and combinations thereof; preferably, the basal medium is a mixture of higher DMEM/F12 and neural basal medium in a ratio of 1:1 (v/v).

12. The method according to any one of claims 1 to 11, wherein one or more selected from the group consisting of serum substitutes, substituted carbon sources, non-essential amino acids, L-glutamine or substitutes thereof, and antibiotics are further added to the medium.

13. The method of claim 12, wherein,

The serum replacement is selected from the group consisting of KOSR, N2, and B27, and combinations thereof; preferably, the serum replacement is a mixture of N2 and B27 in a ratio of 1:1 (w/w);

the alternative carbon source is pyruvic acid, such as sodium pyruvate;

the L-glutamine or the substitute thereof is Glutamax containing L-alanyl-L-glutamine dipeptide ^TM A supplement; and/or

The antibiotic is selected from penicillin, streptomycin or a mixture of penicillin and streptomycin.

14. A method according to any one of claims 1 to 3, wherein the method of preparing PSCs/ics in raw form comprises:

(a) Gene editing primate PSCs by knocking down and/or knocking out one or more associated genes in the cell to reduce the activity of SAH, PRC and/or EZH2 of the PSCs; and

(b) Culturing the genetically engineered cell obtained in step (a) in a medium comprising: TSA at a final concentration of 3-30nM or VPA at a final concentration of 0.25-2mM or NaB at a final concentration of 0.25-2mM, preferably TSA at a final concentration of 3-10nM or VPA at a final concentration of 0.25-1mM or NaB at a final concentration of 0.25-1mM and optionally DZNep at a final concentration of 5-15nM or CPI-1205 at a final concentration of 0.5-2mM, or TSA at a final concentration of 3-10nM or VPA at a final concentration of 0.25-0.5mM or NaB at a final concentration of 0.25-0.5mM and optionally DZNep at a final concentration of 5-80nM, preferably 5-50nM or CPI-1205 at a final concentration of 0.5-5 mM; l-ascorbic acid with a final concentration of 40-70 mug/mL; LIF with a final concentration of 10-30 ng/mL; PD0325901 with a final concentration of 0.5-1.5 mu M; IWR1 or XAV939 at a final concentration of 3-6. Mu.M; wherein the culture medium further comprises:

preferably, the medium contains:

5nM TSA, or 0.5mM VPA, or 0.5mM NaB;50 μg/mL L-ascorbic acid; 20ng/mL LIF;1 μM PD0325901;5 μM IWR1 or 5 μM XAV939; and optionally 10nM DZNep or 1mM CPI-1205; wherein the medium further comprises: (1) 20ng/mL ACTIVIN A or NODAL,1 μ M Y27632, thiazovivin or hydroxyfasudil, and 0.2% (v/v) extracellular matrix; or (2) 20ng/mL ACTIVIN A or NODAL, and 1 μ M Y27632, thiazovivin or hydroxyfasudil; (3) 20ng/mL ACTIVIN A or NODAL, and 0.2% (v/v) extracellular matrix; or (4) 1 μ M Y27632, thiazovivin or hydroxyfasudil, and 0.2% (v/v) extracellular matrix; or (5) 20ng/mL ACTIVIN A or NODAL, or 1 μ M Y27632, thiazovivin or hydroxyfasudil, or 0.2% (v/v) extracellular matrix.

15. The method of any one of claims 1-3, wherein the method of preparing 8CLCs comprises:

(a) Gene editing primate PSCs or primary PSCs/ICLCs by knocking down and/or knocking out one or more associated genes in the cell to reduce the SAH, PRC and/or EZH2 activity of the PSCs;

(b) Culturing the genetically engineered cell obtained in step (a) in a medium comprising: TSA at a final concentration of 10-30nM, or VPA at a final concentration of 0.5-1.5mM or NaB at a final concentration of 0.5-1.5 mM; l-ascorbic acid with a final concentration of 40-70 mug/mL; LIF with a final concentration of 10-30 ng/mL; PD0325901 with a final concentration of 0.5-1.5 mu M; IWR1 or XAV939 at a final concentration of 3-6. Mu.M; and optionally, DZNep at a final concentration of 40-70nM or CPI-1205 at a final concentration of 2-4 mM; and wherein the medium further comprises:

preferably, the medium contains: 20nM TSA, or 1mM VPA, or 1mM NaB;50 μg/mL L-ascorbic acid; 20ng/mL LIF;1 μM PD0325901;5 μM IWR1 or 5 μM XAV939; and optionally 50nM DZNep or 3mM CPI-1205; wherein the medium further comprises (1) 20ng/mL ACTIVIN A or NODAL,1 μ M Y27632, thiazovivin or hydroxyfasudil, and 0.2% (v/v) extracellular matrix; or (2) 20ng/mL ACTIVIN A or NODAL, and 1 μ M Y27632, thiazovivin or hydroxyfasudil; (3) 20ng/mL ACTIVIN A or NODAL, and 0.2% (v/v) extracellular matrix; or (4) 1 μ M Y27632, thiazovivin or hydroxyfasudil, and 0.2% (v/v) extracellular matrix; or (5) 20ng/mL ACTIVIN A or NODAL, or 1 μ M Y27632, thiazovivin or hydroxyfasudil, or 0.2% (v/v) extracellular matrix.

16. The method of any one of claims 1-15, wherein the primate PSC is selected from the group consisting of:

(i) Cells of the ESC and/or ECC series;

(ii) Cells of the iPSC line;

(iii) Cells of the Inner Cell Mass (ICM) of the pre-implantation blastocyst cultured in vitro;

(iv) Cells of the Inner Cell Mass (ICM) of the blastocyst after implantation cultured in vitro;

(v) Cells of embryos from 8 cells (8C) stage to morula stage cultured in vitro.

17. The method of any one of claims 1-16, wherein the primate PSC or primary PSC/ICLC is cultured under one or more conditions selected from the group consisting of: (i) on feeder cells; (ii) on an extracellular matrix without feeder cells; (iii) in suspension without feeder cells; (iv) under hypoxic or normoxic conditions at about 37 ℃; (v) Passaging every 3 to 4 days with single cells at a split ratio of 1:4 to 1:8; (vi) daily medium changes.

18. The method of any one of claims 1-3, further comprising the step of culturing somatic cells in the presence of a SAH/PRC/EZH2 inhibitor, an HDAC inhibitor, and a WNT/β -catenin signaling inhibitor to reprogram the somatic cells to produce primate primary PSC/ICLC.

19. A teratoma produced according to the method of any one of claims 1 and 4-18, and cells isolated from the teratoma.

20. Organoids produced according to the method of any one of claims 2 and 4-18, and cells isolated from organoids.

21. An embryoid body produced by the method of any one of claims 3 and 4-18, and cells isolated from the embryoid body.